MECHANISTIC STUDIES ON NEDD4 FAMILY HECT E3 UBIQUITIN LIGASES

The NEDD4 family HECT (homologous to E6AP C terminus) E3 ligases contain nine enzymes that catalyze the ubiquitination of numerous important proteins including tumor suppressor PTEN and transcription factors Oct4 and EGR2. The NEDD4 ubiquitination enzymes therefore impact many cellular events and need to be tightly controlled. Dysregulation of NEDD4 enzymes is associated with many pathologies like cancer and neurodegenerative diseases. NEDD4 protein architecture includes an N-terminal C2 domain, followed by two to four WW domains and culminating in a C-terminal HECT domain. Previous work from our lab has demonstrated that a linker region between two of the WW domains can undergo an intra-molecular interaction with catalytic HECT domain, serving an auto-inhibitory role. In this thesis, we investigated the molecular mechanisms of this auto-regulatory linker and found that the linker auto-inhibition can be relieved by either linker phosphorylation, which disrupts the linker-HECT interaction. Moreover, we found that linker autoinhibition can be overcome by the NEDD4 family member binding with allosteric activators like NDFIP1 and ubiquitin variants. We have also demonstrated that the NEDD4 family member WWP1 K740N/N745S germline variants, associated with cancer, do not relieve auto-inhibition as suggested in a recent clinical study. Furthermore, we performed substrate screening with a phosphor-mimetic/activated form of the NEDD4 family member WWP2 using protein microarrays. We have identified a group of new potential WWP2 protein substrates from this screening and followed up on three of these, the autophagy receptors NDP52, OPTN and SQSTM1. In vitro and in vivo, we demonstrated that WWP2 ubiquitinates these autophagy receptors and plays an important role in regulating mitophagy. In addition, in this thesis work, we developed two chemical strategies to site-specifically modify proteins of interest for biochemical studies. Firstly, we developed an N-terminal labeling method with NHS ester compounds, which shows high specificity. We applied this strategy to study WWP2 autoubiquitination and E2-E3 molecular interaction. Secondly, we adapted a recently developed technique for chemically-ubiquitinating protein of interest using ubiquitin hydrazide mimics. We expanded its application to other proteins, including ubiquitin-conjugating enzyme E2 and HECT E3 ligases. We applied the generated E2-Ub or E3-Ub as enzyme intermediate state mimics to study the HECT E3 catalytic mechanisms. Our data support a model in which the HECT-Ub intermediate undergoes a conformational switch from an inverted T shape to an L shape, which reduces the E3 affinity for the E2. This model accounts for an efficient turnover process that allows for rapid E2 recycling and enhanced substrate ubiquitination