Characterizing the Cross-Regulatory Complex, OTUB1:E2

Abstract

The ubiquitin system regulates several diverse biological processes in eukaryotes. The process of ubiquitination involves a multienzymatic E1-E2-E3 cascade that serves to activate ubiquitin, conjugate the C-terminus of ubiquitin, and finally to ligate the ubiquitin molecule to a substrate protein via the E3 ligase. Deubiquitinating enzymes (DUBs) reverse these ubiquitin modifications. OTUB1 is a highly expressed DUB that specifically cleaves K48 poly-ubiquitin chains. It helps to regulate ubiquitin concentrations in the cell through its catalytic and non-canonical activities. OTUB1 possesses a unique property among DUBs in that it binds to a subset of ubiquitin conjugating enzymes, E2s, independently of its catalytic activity. By interacting with E2s, a cross-regulation occurs in which the E2s stimulate OTUB1’s isopeptidase activity and, in turn, OTUB1 inhibits the E2’s ability to transfer ubiquitin. Mass spectrometry/proteomics studies revealed that OTUB1 interacts with 7 E2s in cells: UBE2D1, UBE2D2, UBE2D3, UBE2N, UBE2E1, UBE2E2, and UBE2E3. It is unclear whether cross-regulation occurs in vivo. To investigate the mechanism involved in the OTUB1:E2 cross-regulatory complex, I utilized a series of biochemical and kinetic assays. First, I looked at stimulation by determining EC50s and Michaelis Menten kinetic parameters using a FRET assay and quantified the degree of stimulated isopeptidase activity with each E2. I found that binding of an E2 lowers the KM of OTUB1 for K48 diUb, at a broad range of stimulation depending on the E2, with little to no change in the kcat. This result indicates that the E2 increases OTUB1’s affinity for K48 diUb. I used ITC to confirm this, my results indicate that the interactions between OTUB1 and the E2 produces an increase in OTUB1’s affinity for K48 chains. I next sought to characterize OTUB1’s inhibitory effects on E2 ubiquitin conjugation. Similar to previous studies, OTUB1 blocks both UBE2N and UBE2D proteins from free chain poly-ubiquitination. Unlike UBE2D or UBE2N where OTUB1 inhibits their poly-ubiquitination activity, OTUB1 predominantly serves to stop autoubiquitylation of UBE2E enzymes. Further in vivo studies revealed that autoubiquitylation of UBE2E1 targets it from proteasomal degradation. By inhibiting autoubiquitylation, OTUB1 stabilizes UBE2E1; a novel role for OTUB1.