Non-proteolytic ubiquitination of OTULIN regulates NF-κB signaling pathway

Abstract

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.

Nuclear soluble cGAS senses DNA virus infection

“…Western blotting and immunoprecipitation were performed as described in a previous study (Zhao et al, 2019). Briefly, samples (10–15□μl) were loaded into Mini-Protean TGX Precast Gels, 15 well (Bio-Rad, # 456-103), and run in 1 × Tris/Glycine/SDS Buffer (Bio-Rad, # 161-0732) for 60□min at 140□V.…”

Linear Ubiquitin Code: Its Writer, Erasers, Decoders, Inhibitors, and Implications in Disorders

“…The OTULIN-mediated suppression of hepatocyte apoptosis plays a crucial role in liver pathogeneses, such as hepatitis, fibrosis, and hepatocellular carcinoma [96]. TRIM32, a RING-type E3, conjugates the K63-linked ubiquitin chain to OTULIN to interfere with its interaction with HOIP in human embryonic kidney (HEK) 293 cells [97], and the interaction between TRIM32 and SNX27, which regulates endosome-to-plasma membrane trafficking, has been confirmed [98]. Thus, OTULIN is a crucial pathophysiological regulator in the linear ubiquitin code.…”

Ubiquitin-dependent and -independent functions of OTULIN in cell fate control and beyond

“…TRIM32 interacts with the OTULIN OTU domain and conjugates non-proteolytic Lys63-linked poly-Ub at two lysine residues (Lys64 and Lys66) in the vicinity of the PIM domain (Fig. 1a ), thereby reducing the HOIP interaction and enhancing TNFα-induced NF-κB activation [ 48 ]. Importantly, stimulation with TNFα increased the OTULIN–TRIM32 interaction, indicating that TRIM32-mediated ubiquitination contributes to the control of inflammatory signaling [ 48 ].…”

“…1a ), thereby reducing the HOIP interaction and enhancing TNFα-induced NF-κB activation [ 48 ]. Importantly, stimulation with TNFα increased the OTULIN–TRIM32 interaction, indicating that TRIM32-mediated ubiquitination contributes to the control of inflammatory signaling [ 48 ].…”