Characterization of the Role of Tandem Ubiquitin Binding Motif (tUBM) of E3 Ligase HUWE1

As an E3 ligase, HUWE1 regulates various cellular processes including protein stability, DNA repair, and transcription regulation through ubiquitinating its substrate proteins. Mutation and deregulation of HUWE1 are associated with many diseases including neuronal disorders and cancers. However, the structure and function of HUWE1 protein has not been fully revealed. The tandem ubiquitin binding motif (tUBM), a novel domain of HUWE1, hypothetically modulates E3 ligase activity of HUWE1 through recognizing ubiquitin or ubiquitin chains. HUWE1 R2981H missense mutation within the tUBM domain was reported as one of the causes of X linked Intellectual Disability thus highlighting the importance of the tUBM domain. Therefore, tUBM domain was characterized in this project and the main findings were that: (1) tUBM interacts all eight types of ubiquitin di-chains; (2) the tUBM domain could modulate HECT domain activity without altering its Ub linkage preference in vitro, and (3) the HECT domain was found to be capable of synthesizing polyUb chains not only using linkage specific monoUb, but also using different diUbs as reactants. Additionally, two ubiquitin variants being developed to target UBM1 were discovered to be capable of modulating tUBM-HECT catalysis in a dose dependent manner. Therefore, the novel HUWE1 tUBM domain may serve as a Ub chains enrichment module and thus be targeted to modulate HUWE1 function.