Ubiquitin‐Activated Interaction Traps ( UBAIT s) identify E3 ligase binding partners

Abstract

We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (UbiquitinActivated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1-and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ-a histone protein involved in DNA repair-as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.

Extracellular ISG15 Signals Cytokine Secretion through the LFA-1 Integrin Receptor

“…To identify the receptor we utilized an ISG15 UBAIT ( Ub iquitin- A ctivated I nteraction T rap) (O’Connor et al, 2015). UBAITs are fusions of a protein of interest (in this case ISG15) to a short flexible linker and ubiquitin (Figure 3A).…”

“…While first described as reagents for identification of ubiquitin ligase substrates (O’Connor et al, 2015), UBAITs, as shown here, can be used for characterizing the interactome of virtually any protein of interest. UBAITs were previously used as intracellular reagents (Byun et al, 2017; O’Connor et al, 2015). In contrast, the ISG15-UBAIT, charged in vitro , was used as an extracellular reagent to identify cell surface interacting proteins.…”

“… A. UBAITs are fusion proteins consisting of an affinity tag, a protein of interest, a short linker, and a C-terminal ubiquitin moiety (O’Connor et al, 2015). UBAITs can be activated by the E1 ubiquitin-activating enzyme and then transferred to an E2 protein.…”

E3 Ubiquitin ligase RNF126 regulates the progression of tongue cancer

“…First, the substrates of RNF126, like p21 14, Bag6 15, and EGFR 9 are involved in oncogenesis. It promotes cell proliferation partially through degrading p21 14.…”

Systematic approaches to identify E3 ligase substrates

“…A more recently developed trapping approach uses E3-ubiquitin fusion proteins (ubiquitin-activated interaction traps, UBAITs) to covalently capture proteins that interact with the E3 in an E1- and E2-dependent manner. This technique was successfully identified binding partners of both HECT (Rsp5 and Itch) and RING (Psh1, RNF126 and RNF168) E3 ligases [95]. …”