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Non-proteolytic ubiquitination of OTULIN regulates NF-κB signaling pathway Free
Mengmeng Zhao1,† , Kun Song1,† , Wenzhuo Hao1,† , Lingyan Wang1 , Girish Patil1 , Qingmei Li1,2 , Lingling Xu1 , Fang Hua1 , Bishi Fu3 , Jens C. Schwamborn4 , Martin E. Dorf5 , Shitao Li1,*
1Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA
2Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China
3State Key Laboratory of Virology, Medical Research Institute, Wuhan University, Wuhan, China
4Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Luxembourg City, Luxembourg
5Department of Immunology, Harvard Medical School, Boston, MA 02115, USA
These authors contributed equally to this work.
*Correspondence to:Shitao Li , Email:sli38@tulane.edu
J Mol Cell Biol, Volume 12, Issue 3, March 2020, 163-175,  https://doi.org/10.1093/jmcb/mjz081
Keyword: NF-κB, LUBAC, TNF, linear ubiquitination, TRIM, proteomics

NF-κB signaling regulates diverse processes such as cell death, inflammation, immunity, and cancer. The activity of NF-κB is controlled by methionine 1-linked linear polyubiquitin, which is assembled by the linear ubiquitin chain assembly complex (LUBAC) and the ubiquitin-conjugating enzyme UBE2L3. Recent studies found that the deubiquitinase OTULIN breaks the linear ubiquitin chain, thus inhibiting NF-κB signaling. Despite the essential role of OTULIN in NF-κB signaling has been established, the regulatory mechanism for OTULIN is not well elucidated. To discover the potential regulators of OTULIN, we analyzed the OTULIN protein complex by proteomics and revealed several OTULIN-binding proteins, including LUBAC and tripartite motif-containing protein 32 (TRIM32). TRIM32 is known to activate NF-κB signaling, but the mechanism is not clear. Genetic complement experiments found that TRIM32 is upstream of OTULIN and TRIM32-mediated NF-κB activation is dependent on OTULIN. Mutagenesis of the E3 ligase domain showed that the E3 ligase activity is essential for TRIM32-mediated NF-κB activation. Further experiments found that TRIM32 conjugates polyubiquitin onto OTULIN and the polyubiquitin blocks the interaction between HOIP and OTULIN, thereby activating NF-κB signaling. Taken together, we report a novel regulatory mechanism by which TRIM32-mediated non-proteolytic ubiquitination of OTULIN impedes the access of OTULIN to the LUBAC and promotes NF-κB activation.