Proteases to the rescue: Decoding de-ubiquitination in cancer

Proteolysis is fundamental to virtually all cellular processes such as DNA damage repair, cell cycle regulation, and the immune response. Ubiquitination is a key modification for regulating the activity and levels of proteins in eukaryotes. Ubiquitin specific proteases (USPs) can rescue proteins from destruction by the proteasome by reversing ubiquitination. The human genome encodes 56 USPs and dysregulated de-ubiquitination is associated with cancer, neurodegenerative disorders, and the host's response to infection. Modulation of USP function offers valuable avenues for therapeutic intervention and biotechnological applications. However, it is currently unclear how these proteases specifically recognize their substrates: Thousands of human proteins have been shown to be ubiquitinated and the largest binding pocket in USPs accommodates the ubiquitin marker common to most substrates. This not only has major implications for our fundamental understanding of how these proteases function but also for drug discovery efforts.
The main aims of this project are to (1) determine the key structural features that allow a USP to recognize its substrates and (2) to utilize this knowledge to modulate USP function in cancer cells. We will focus on USP11 that associates with homologous recombination proteins and is a key regulator of DNA damage repair reversing ubiquitination of several proteins of therapeutic interest such as p21, histones and p53. Depletion of USP11 has been shown to be effective in targeting DNA damage response (DDR) pathway deficient tumours such as those that are BRCA2 defective. Moreover, silencing of USP11 is effective in killing clinically relevant platinum-resistant cancer cells and can inhibit pancreatic cancer cell survival. In addition, USP11 may also be of interest as a target in viral infection and neurodegenerative disease. The project will consist of the following parts:
(a) Production of available and novel ubiquitin fusion substrates relevant to cancer that will be used to probe USP11 specificity in binding and enzymatic assays using established protocols. (b) Structure determination of a USP11-substrate complex using state-of-the-art structural biology techniques including X-ray crystallography or cryo-electron microscopy to gain detailed molecular insight into the interactions. (c) Mutagenesis of identified binding sites and proof-of concept studies to test the impact of blocking these in cellular assays on cell viability. The project builds on existing expertise and exciting preliminary data available in the lab.
Together, the project will offer skill development in an interdisciplinary setting including protein biochemistry, fluorescent assays, structural biology and cell biology and will deliver novel insights into USP11 function, structure and inhibition providing the basis for manipulation of protein degradation to ultimately target cancer cells.