The Development and Characterization of Ubiquitin Variants as Molecular Probes of E3 Ligase HUWE1

Abstract

Mutations in the ubiquitin E3 ligase HUWE1 have been linked to XLID. However, the large size of HUWE1 poses challenges for studying the mechanisms and functions of its domains using current technologies. In this study, two Ubiquitin Variants (UbVs) were developed via a collaboration with Dr. Wei Zhang’s lab (University of Guelph) to bind to the UBM1 and HECT domains of HUWE1 specifically, and their targeted binding affinities and cross-interactions were investigated. Additionally, the interactions of the HUWE1 protein with both UbVs and a fusion of these two UbVs were further validated in cells. Lastly, the effect of UbVs on endogenous HUWE1 was assessed by comparing HUWE1 substrate levels in the proteasome inhibitor MG132 treated and untreated cells. Our findings reveal that both HUWE1 UbVs interact strongly with their respective targeted domains; both HUWE1 UbVs and the fusion UbV interact with HUWE1 protein and modulate its function in cells.