Studies on AUF1-regulated deubiquitinase complexes: USP1/UAF1, USP12/UAF1 and USP46/UAF1

Abstract

Deubiquitinases (DUBs) regulate a wide range of cellular processes and are potential targets for cancer therapy. Our knowledge on the biology of these enzymes is still limited. We focused on three human DUBs (USP1, USP12 and USP46) that share a common regulatory mechanism: they are activated by UAF1. We have explored several aspects of the function and regulation of these three DUBs to clarify standing controversies and to uncover novel details. Our results show that phosphorylation of USP1 at serine 313 is not necessary for UAF1 binding, neither for its catalytic activity. We also provide evidence that USP1 autocleavage occurs in cis. We map the UAF1-binding site of USP1, USP12 and USP46 to their ¿Fingers¿ subdomain, and contribute to identify critical residues for the interaction between these DUBs and UAF1. We show for the first time that cancer-related mutations in USP1 can alter its function and regulation. Regarding the potential role of the USP1/UAF1 complex as a target in cancer, we observed overexpression of USP1 and UAF1 in pancreatic tumors and derived cell lines, and our data suggest that the USP1 inhibitor ML323 can increase oxaliplatin sensitivity in a relatively resistant pancreatic cancer cell line. In this Thesis we also significanlty expand the repertoire or potential USP1 substrates. By applying a novel approach, termed bioUB, and label-free quantification (LFQ)-based proteomics, we have identified 10 proteins whose ubiquitination level increases when USP1 is silenced. Finally, regarding USP12 and USP46, our data suggest that these enzymes may differently bind to their common cofactor WDR20, and to at least another interactor (DMWD). This differential binding appears to be mediated by the presence/absence of a 4 amino acid motif (MEIL) at their N-terminus, and may contribute to explain the previously observed functional differences between these two highly similar enzymes.