2.2.5. LUBAC-Mediated Regulation of Interferon Signaling

In innate immune responses, PAMPs are recognized by host pattern-recognition receptors, and then activate the NF-κB and type I interferon (IFN) production pathways. In the course of the IFN antiviral pathway, the phosphorylation of transcription factors, such as interferon regulatory factor 3 (IRF3), by TANK-binding kinase 1 (TBK1) and IKKε causes the transcription of IRF3-target genes, including *IFN*β, *ISG15*, and *ISG56*. [64]. Although various E3s are known to regulate the antiviral pathway [65], whether LUBAC activates or suppresses the type I IFN production pathway remains controversial. LUBAC and the linear ubiquitination activity reportedly down-regulate the type I IFN production pathway [66,67]. STAT1 is linearly ubiquitinated at the K511 and K652 residues by LUBAC, which inhibits binding to the type I IFN receptor, IFNAR2, and the linear ubiquitination of STAT1 is removed by OTULIN [68]. By contrast, virus infection induces the enhanced LUBAC-mediated NF-κB activation, which in turn, inhibits IFN-STAT1 signaling.

However, some studies suggested that the LUBAC activity is necessary for the TLR-mediated IRF3 activation [69–71], and LUBAC is reportedly indispensable for the TNF-induced TBK1 and IKKε activation and prevention of cell death [72]. We showed that the LPS-, poly(I:C)-, and SeV-mediated IFN production pathway was impaired in *HOIP*-deficient mouse embryonic fibroblasts and human Jurkat T-lymphoblasts, and LUBAC inhibitors, HOIPINs, suppressed the antiviral pathway through the reduced activation of TBK1 and IRF3, supporting the positive function of LUBAC in this pathway [73]. Further studies are required to clarify the function of LUBAC in the IFN antiviral pathway.

### 2.2.6. Involvement of Linear Ubiquitination in Selective Autophagy

Damaged organelles and invading pathogens are selectively degraded through the autophagy pathway [74]. The selective autophagy against intracellular bacteria is referred to as xenophagy. For instance, invaded *Salmonella* replicates within the host-derived membrane vacuole, and the rupture of the vacuole causes ubiquitination and autophagy [75]. The ubiquitinome analysis showed that *Salmonella* infection promotes CDC42 and LUBAC activities with the enhanced NF-κB activity through linear ubiquitination in human colon cancer HCT116 cells and HeLa cells [76]. In addition to several E3s, such as Parkin, Smurf1, RNF166, ARIH1, and LRSAM1, LUBAC indeed restricts *Salmonella* proliferation through linear ubiquitination, which functions to recruit optineurin (OPTN) and NEMO to induce xenophagy and activate NF-κB, respectively [77,78]. Moreover, a linear ubiquitin-specific DUB, OTULIN, antagonizes LUBAC function in the xenophagy of *Salmonella* [79]. These results indicate that linear (de)-ubiquitination is indispensable for the regulation of bacterial pathogenesis and selective autophagy.

### **3. Erasers of the Linear Ubiquitin Code**

DUBs function in the biosynthesis, recycling, editing, and cleavage of ubiquitin linkages, and consequently maintain the intracellular free ubiquitin pool [80,81]. The human DUBs are classified into seven subfamilies: ubiquitin-specific protease (USP; 54 members), ubiquitin C-terminal hydrolase (UCH; 4 members), ovarian tumor protease (OTU; 16 members), Josephins (4 members), motif interacting with ubiquitin (MIU)-containing novel DUB (MINDY; 4 members) [82], zinc finger with UFM1-specific peptidase domain protein (ZUFSP; 2 members) [83], and JAB1/MPN/MOV34 metalloenzymes (JAMM/MPN+; 16 members) [80,81]. The USP, UCH, OTU, Josephin, MINDY, and ZFUBP DUBs belong to the Cys protease family, whereas the JAMM/MPN+ family proteins are zinc metalloproteases. Among the DUBs, OTULIN (OTU family), CYLD (USP family), and A20 (OTU family) (Figure 3) are well known DUBs that regulate the LUBAC-mediated NF-κB signaling pathway [84]. In this chapter, we introduce OTULIN and CYLD as "erasers" of the linear ubiquitin code, and classify A20 as a "decoder" (see Section 4.2).

**Figure 3.** DUBs involved in the regulation of the linear ubiquitin code. Domain structures and functions of LUBAC-regulating factors, such as OTULIN, CYLD-SPATA2, and A20, are shown. PIM, PUB domain-interacting motif; OTU, ovarian tumor protease; CAP-Gly, cytoskeleton-associated protein Gly-rich domain; B box, B-box-type zinc finger domain; PUB, PNGase/UBA or UBX; PHD, plant homeodomain; ZF, zinc finger. Phosphorylation sites are denoted by encircled Ps, and caspase 8 and MALT1 cleavage sites are also indicated.
