Identification of linear ubiquitination substrates with UBAN domain probe3 / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE In this research, the ubiquitin binding in ABIN and NEMO proteins (UBAN) domain of NF-kB essential modulator (NEMO) was used as a protein probe to screen potent linear ubiq-uitination substrates. Adopting the proteomics strategy, the putative substrates could be enriched and identified. This work will greatly expand our knowledge on the molecular mechanism of linear ubiquitina-tion regulation in various biological events. METHODS ® The glutathione S-transferase (GST)-UBAN protein was overexpressed by Escherichia coli and connected with glutathione agarose beads by GST tag to be used as the UBAN probe. Then, the function of the probe was verified in linear ubiquitination chain binding ability test by immunoprecipitation and Western blotting in 293T cells. Using the UBAN probe.linear ubiquitination substrates were enriched and identified by mass spectrometry in 293T cell lysate and analyzed by bioinformatics. Among them, the potential linear ubiquitination substrates Aurora kinase A (Aurora-A) and linear ubiquitin chain assembly complex (LUBAC) were co-transfected in 293T cells and the linear ubiquitination chain binding ability of Aurora-A was identified by Western blot. ® LUBAC was interfered in with siRNA and then synchronized in HeLa cells. The regulation of linear ubiquitination on the biological function of potential substrate Aurora A was observe by Western blotting. RESULTS (1) Western blotting results suggested that the UBAN probe could bind to the linear ubiquitin chain on NEMO, which confirmed the success of the system. ® Including Aurora-As a total of 403 proteins were identified as potential substrates of linear ubiquitination by mass spectrometry. The ability to bind to the linear ubiquitination chain was confirmed by immunoprecipitation between Aurora-A and the linear ubiquitination chains. ® Western blotting results showed that compared with the control group, Aurora-A phosphorylation level was up-regulated, which meant the occurrence of self-activation after knockdown of LUBAC. CONCLUSION The probe constructed with the UBAN domain can efficiently capture and enrich linear ubiquitination substrates. And the self-activation of Aurora-A may be regulated by linear ubiquitination. Significantly, the strategy of identifying the linear ubiquitination substrates established in this paper provides a novel idea and method for further exploration of biological functions of linear ubiquitination.