SPOP-mediated Non-degradative Ubiquitination / 中国生物化学与分子生物学报

Protein post-translational modification is a precondition guaranteeing normal exertion of protein functions. Ubiquitination is an important post-translational modification that maintains normal protein levels and activity. Numerous researches show that the E3 ubiquitin ligase speckle-type POZ protein (SPOP) displays mutations in many tumors and genetic diseases. Mainly concentrated in the MATH structural domain that recognizes substrates, these mutations influence the binding between SPOP and substrates, and further influence their protein levels, positioning and activities, thus disturbing the normal physiological functions. Wild-type SPOP binds the substrates, most of which enter the proteasome pathway for decomposition after being ubiquitinated by SPOP, but some substrates are also influenced functionally. Herein we review the ubiquitination types and functions of SPOP substrates, including the ubiquitin-proteasome system (UPS), structure, functions and molecular pathways of SPOP, and non-degradative ubiquitinated modification of SPOP. The emphasis will be laid on the molecular mechanisms of the signaling pathways mediated by the three non-degradative substrates of SPOP, that is, myeloid differentiation primary response gene 88 (MyD88)-mediated NF-κB pathway, X-chromosome silence signal pathway of histone macroH2A1 (macroH2A. 1 histone, macroH2A1), and inverted formin 2 (INF2)mediated chondriokinesis pathway, in inhibiting tumorigenesis and development. We expect to provide a new perspective for precise targeted therapies of tumors.

Mechanism of Effect of Ubiquitination Modification Mediated by E3 Ubiquitin Ligase in Inflammatory Bowel Disease / 胃肠病学

Ubiquitination is the important type of post-translational modification of proteins, and its main effects include protein degradation and functional regulation. Studies have shown that abnormal ubiquitination is involved in the development and progress of inflammatory bowel disease (IBD), which makes some ubiquitinases and their antibodies having the potential to be important markers for clinical diagnosis and evaluation of severity of Crohn's disease (CD). This article reviewed the research on mechanism of effect of ubiquitination modification mediated by E3 ubiquitin ligase in IBD.

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

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.