Abstract
Itch is an E3 ubiquitin-protein ligase that is an important negative regulator of inflammatory signalling, the viral immune response, and controls aspects of cellular differentiation and membrane-receptor sorting. Through these pathways, dysregulation of Itch results in cancer and a severe hereditary autoimmune disease.
Itch is a multi-domain protein with E3 ubiquitin-ligase activity. Itch contains a C2 domain to target its activity to membranes and a proline-rich region with an unknown function. Itch interacts with many proteins via its four WW domains, selecting these proteins for ubiquitylation by the HECT domain. Itch-dependent ubiquitylation is a crucial part of many cellular signalling cascades. This is a highly regulated process that is not well understood.
WW domains interact with proline-rich motifs of substrate proteins, however little is known about how multiple WW domains work in tandem to recruit substrates and adapter proteins. The four WW domains of Itch function to specifically recruit both substrates and adapter proteins. I wished to investigate the specific interactions made by the isolated WW domains of Itch, how the multiple WW domains work in tandem, and if avidity plays a role in substrate recruitment.
The catalytic HECT domain, with E3 ubiquitin-protein ligase activity, must be regulated in order for signalling to occur at the appropriate time. I aimed to investigate if the proline-rich region of Itch competes with substrate binding to the WW domains, therefore affecting ubiquitylation of these substrates. Finally, I set out to explore the effect of substrate binding on Itch-dependent ubiquitylation.
I have used bioinformatic analysis, GST pull-down assays,bio-layer interferometry, and in vitro ubiquitylation assays in order to elucidate the binding properties of the WW domains and how the HECT domain is controlled using two model substrate proteins, RNF11 and NDFIP1.
I have shown that RNF11 and NDFIP1 have specific and preferential interactions with the WW domains of Itch. Additionally, the experiments revealed that the two non-canonical proline-rich motifs of RNF11 contribute to the interaction with the WW domains of Itch. These experiments indicate there are multiple interactions between the WW domains and the proline-rich motifs of both model substrates, therefore, avidity does play a role in substrate recruitment by Itch. Contrary to the initial hypothesis, the protein-protein interactions made by the WW domains are not inhibited by the proline-rich region of Itch. However, the experiments in this study show that ubiquitylation is affected by this proline-rich region, leading us to suspect an alternative mechanism of control. Additionally, these experiments have shown that binding of substrates to the WW domains promotes the E3 activity of Itch, possibly by relieving an auto-inhibitory mechanism. This implies other adapters and substrates of Itch may also activate its E3 activity.
Itch is an important regulator of many cellular processes, acting as a protein-protein interaction hub, binding to many different substrate proteins via its WW domains. Progress has been made in understanding the interactions made by the WW domains of Itch and how ubiquitylation by the HECT domain is controlled.