Enabling Protein Degradation Drug Discovery

Deconjugating enzymes (DCEs) are proteases that process ubiquitin or ubiquitin-like gene products, reverse the modification of proteins by a single ubiquitin or ubiquitin-like protein (UBL) and remodel polyubiquitin (or poly-UBL) chains on target proteins (Reyes-Turcu et al., 2009). The deubiquitylating – or deubiquitinating – enzymes (DUBs) represent the largest family of DCEs and regulate ubiquitin dependent signalling pathways. The activities of the DUBs include the generation of free ubiquitin from precursor molecules, the recycling of ubiquitin following substrate degradation to maintain cellular ubiquitin homeostasis and the removal of ubiquitin or ubiquitin-like proteins (UBL) modifications through chain editing to rescue proteins from proteasomal degradation or to influence cell signalling events (Komander et al., 2009). There are two main classes of DUB, cysteine proteases and metalloproteases. A20 is a cysteine protease and is a member of the OTU (ovarian tumour) superfamily of proteins (Balakirev et al., 2003). Cloning of the human gene was first described by Opipari et al. (1990). A20, or TNFAIP3, is a ubiquitin-editing enzyme that contains both ubiquitin ligase and deubiquitylase activities. The N-terminal domain of A20 functions as a deubiquitylating enzyme by removing K63-linked ubiquitin chains from receptor-interacting protein (RIP) essential mediator of the proximal TNF receptor-1 (TNFR1) signalling complex. The C-terminal domain of A20, composed of 7 C2/C2 zinc fingers, then functions as a ubiquitin ligase by polyubiquitylating RIP with K48- linked ubiquitin chains, thereby targeting RIP for proteasomal degradation (Wertz et al., 2004). Literature evidence suggests a mechanism of action for A20 in the inhibition of inflammatory signalling pathways.Research showed that A20 inhibits the E3 ligase activities of TRAF6, TRAF2, and cIAP1 by antagonizing interactions with E2 ubiquitin-conjugating enzymes Ube2N and Ube2D3. A20, together with the regulatory molecule TAX1BP1, interacted with Ube2N and Ube2D3 and triggered their ubiquitylation and proteasome-dependent degradation (Shembade et al., 2010). More recently, studies have highlighted the clinical and biological importance of A20. Human genetic research has shown strongly linked polymorphisms and mutations in the gene encoding A20 to inflammatory, autoimmune and malignant diseases. Furthermore, studies in gene-targeted mice have revealed that A20 regulates multiple immune cell functions and prevents experimental diseases that closely mimic human conditions. These studies reveal the importance of A20-mediated regulation of ubiquitin-dependent signalling in human disease (Ma and Malynn, 2012).