*3.2. CYLD*

CYLD, a USP family DUB (Figure 3), was initially identified as a cylindromatosis tumor suppressor gene in humans [99]. CYLD downregulates the NF-κB activation pathway by hydrolyzing K63-linked ubiquitin chains [100,101]. The genetic deficiency in *CYLD* causes trichoepithelioma. Importantly, CYLD e fficiently hydrolyzes the K63-linked ubiquitin chain and the linear ubiquitin chain, but not the K48-chain [102], and thus regulates innate immune signaling [103]. We and another group showed that the catalytic activity of CYLD is indispensable for the downregulation of LUBAC-mediated NF-κB activation [104,105]. Furthermore, we revealed the structural bases of the CYLD USP domain recognition of either K63 or linear diubiquitin [106], and the interaction of a potential CYLD inhibitor, subquinocin [107]. We further identified that mind bomb homolog 2 (MIB2) is an E3 that leads to the proteasomal degradation of CYLD, and therefore MIB2 a ffects LUBAC-mediated NF-κB activation [108]. Importantly, the USP domain of CYLD binds to the PUB domain of SPATA2, and the PIM in SPATA2 associates with the PUB domain of HOIP (Figure 3) [26–29]. Recently, a familial variant of *CYLD* with the M719V missense mutation was identified in the induction of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), with enhanced K63-deubiquitinating activity and NF-κB suppression [109]. Moreover, this M719V variant of CYLD impairs autophagosome maturation and increases the cytosolic localization of TDP-43. Thus, the up-regulation of CYLD activity is associated with neurodegenerative diseases, whereas the down-regulation of *CYLD* causes cancers.

### **4. Decoders of the Linear Ubiquitin Code**

In the ubiquitin code, various types of ubiquitin chains serve as sca ffolds to recruit their specific binding proteins, and subsequently, these locally concentrated proteins are responsible for the cellular functions of the ubiquitin code. Therefore, the ubiquitin chain-specific binding proteins are referred to as "decoders". In the linear ubiquitin code, several protein motifs, such as the UBD in ABIN proteins and the NEMO (UBAN) domain [110], the NZF domain in HOIL-1L [30], and the 7th zinc finger domain in A20 (A20 ZF7) [104,111], have been identified as linear ubiquitin chain-specific binding domains [112]. In this chapter, we summarize the structures and functions of linear ubiquitin-binding proteins.

### *4.1. UBAN Domain-Containing Proteins: NEMO, OPTN, and ABINs*

The UBAN domain forms a homodimeric coiled-coil structure, and is present in proteins such as NEMO, OPTN, and A20-binding inhibitors of NF-κB (ABIN), including ABIN-1, ABIN-2, and ABIN-3 [110]. Although the K63-linked and linear diubiquitins adopt similar conformations, the NEMO UBAN domain shows approximately 100-fold higher a ffinity to the linear ubiquitin chain than the K63-chain. The hydrophobic patches centered at Ile44 and Phe4 of the distal and proximal parts of linear ubiquitin, respectively, are crucial for the interactions with the UBAN domain [113,114]. The NEMO-UBAN domain is critical for the recruitment of the IKK complex onto the linear ubiquitin chain, which induces the canonical NF-κB activation [32]. Missense mutations in the NEMO-UBAN domain in humans cause X-linked anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) [115,116]. Therefore, the linear ubiquitin-binding function of NEMO is indispensable for homeostasis.

OPTN, initially identified as a gene responsible for primary open-angle glaucoma (POAG) [117], has a similar domain organization to that of NEMO, although it does not interact with IKK <sup>α</sup>/β. OPTN is a multifunctional protein that participates in the regulation of the NF-κB and antiviral signaling pathways, vesicular transport, and selective autophagy, such as mitophagy and xenophagy [118]. In 2010, reports demonstrated that genetic mutations in *OPTN* are associated with ALS [119,120], and include an E478G missense mutation in the UBAN domain of human OPTN. We analyzed the e ffects of the POAG- and ALS-associated mutants of OPTN on the LUBAC- and TNFα-mediated NF-κB activation in HEK293T cells, and showed that the ALS-associated OPTN mutants lost their ability to suppress NF-κB activation, mainly due to the dysfunction in the UBAN domain of OPTN [121]. The OPTN-UBAN domain strongly bound linear ubiquitin, with a Kd value of 1.0 μM, and the crystal structure of the OPTN-UBAN domain complexed with linear ubiquitin revealed that it shares a similar architecture with that of NEMO [121]. Thus, linear ubiquitin binding by OPTN regulates NF-κB activation and apoptosis, and consequently suppresses ALS.

ABINs were originally identified as inhibitors of NF-κB signaling, and they regulate multiple signal transduction pathways, apoptosis, virus replication, and cancer progression [122]. At present, the crystal structures of the UBAN domains in ABIN-1 and -2 with linear ubiquitin have been solved, and they basically adopt conformations similar to those in NEMO and OPTN [123,124]. Therefore, the ABINs are potential decoders of the linear ubiquitin code, through linear ubiquitin-binding by their UBAN domains.
