**1. Introduction**

Ubiquitin, a 76-residue (8.6 kDa) small globular protein, is evolutionally conserved in most eukaryotes. Ubiquitin functions as a spatiotemporal-specific post-translational modifier. In most cases, ubiquitin is covalently conjugated to the ε-NH2 group of Lys in the targeted proteins via an isopeptide bond [1]. In particular cases, ubiquitin is conjugated to the N-terminal α-NH2-group of Lys-less proteins and internal Ser, Thr, and Cys residues [2,3]. The human ubiquitin system is composed of two ubiquitin-activating enzymes (E1), ~40 ubiquitin-conjugating enzymes (E2), >600 ubiquitin ligases (E3), and ~100 deubiquitinating enzymes (DUBs). Among them, the E3s play crucial roles in recognizing and conjugating one or more ubiquitins to substrates, and are classified into the homologous to the E6AP carboxyl terminus (HECT)-type, the really interesting new gene (RING)-type, and the RING-in between-RING (RBR)-type [4]. In the human genome, most of the E3s are RING-type [5], and limited numbers of HECT-type (28 members) and RBR-type (14 members) E3s have been identified.

Protein ubiquitination regulates numerous cellular functions, including proteasomal degradation, membrane trafficking, DNA repair, and signal transduction, through the conjugation of one or

more ubiquitins to substrates [1]. The conjugation of monoubiquitin and multiple-monoubiquitins to substrates is principally involved in membrane tra fficking and endocytosis. The isopeptide bond-linked ubiquitination is mediated via seven internal Lys residues (K6, K11, K27, K29, K33, K48, and K63). Among them, the K48-linked polyubiquitin chain, which is the most common, serves as a typical proteasomal degradation signal, and the K63-linked polyubiquitin chain, the second most predominant linkage, is involved in non-proteasomal functions, such as signal transduction and DNA repair [1,6]. In addition to these Lys-linked polyubiquitin chains, the N-terminal Met1(M1)-linked linear polyubiquitination is specifically generated by the E3 complex, named the linear ubiquitin chain assembly complex (LUBAC). LUBAC functions in the regulation of the innate and acquired immune responses and anti-apoptosis [7]. In addition to the mammalian LUBACs, an ortholog of a HOIP subunit of LUBAC, named LUBEL, was identified in *Drosophila*, and it also catalyzes linear ubiquitination upon heat shock [8]. These findings indicated that linear ubiquitination is evolutionally conserved to maintain cellular homeostasis. In addition to the homotypic polyubiquitin chains, heterogeneous complex-types of polyubiquitin chains, such as mixed, hybrid, and branched ubiquitin chains, have also been identified. Furthermore, specific residues of ubiquitin are chemically modified, by phosphorylation, acetylation, and ADP-ribosylation, and these modifications regulate the pathophysiological functions of ubiquitination. These diverse ubiquitin linkages exhibit multiple functions in a system called the "ubiquitin code" [6], in which E1, E2, and E3 function as "writers", DUBs are "erasers", and ubiquitin-binding proteins serve as "decoders". In this review, we focus on the structures, catalytic mechanisms, inhibitors, and pathophysiological functions of the LUBAC-mediated "linear ubiquitin code", revealed by studies using human cell lines, diseases, and phenotypes of genetically deficient mice.

### **2. LUBAC: The Only Writer of the Linear Ubiquitin Code**

### *2.1. Structure and Catalytic Mechanism of LUBAC*

Mammalian LUBAC, a ~600 kDa complex composed of the HOIL-1L (also known as RBCK1) [9], HOIP (RNF31, ZIBRA, and PAUL) [10], and SHARPIN [11–13] subunits, is the sole E3 that generates the N-terminal M1-linked linear polyubiquitin chain, using the E2s UBE2L3 (UbcH7) and UbcH5s [14,15]. LUBAC subunits contain multiple domains (Figure 1). Although the detailed architecture of LUBAC has not been solved, the ubiquitin-like (UBL) domains in SHARPIN and HOIL-1L bind to the ubiquitin-associated (UBA)1 and UBA2 domains, respectively, in HOIP [16]. Moreover, the LUBAC-tethering motifs (LTMs) in HOIL-1L and SHARPIN associate with each other to form a globular domain [16]. HOIL-1L and HOIP possess RBR-type E3 motifs. The RBR-type E3 family members reportedly generate polyubiquitin chains through a unique RING-HECT-hybrid reaction [17,18]. During the course of linear ubiquitination, the RING1 domain in HOIP binds a ubiquitin-charged E2. Subsequently, the donor ubiquitin is transiently transferred to the active Cys885 in the RING2 domain of HOIP via a thioester-linkage. Finally, the donor ubiquitin is conjugated to an acceptor ubiquitin, which is captured in the C-terminal linear ubiquitin chain determining domain (LDD) of HOIP, to specifically generate a linear ubiquitin chain [19–22]. In contrast to HOIP, the RBR domain in HOIL-1L uniquely catalyzes the oxyester-bond monoubiquitination of Ser/Thr residues through the active Cys458 [23].

**Figure 1.** Domain structure and functional regions of the LUBAC subunits, HOIL-1L, HOIP, and SHARPIN. LTM, LUBAC-tethering motif; UBL, ubiquitin-like; NZF, Npl4-type zinc finger; RING, really interesting new gene; IBR, in-between RING; PUB, PNGase/UBA or UBX; ZF, zinc finger; UBA, ubiquitin-associated; LDD, linear ubiquitin chain determining domain; PH, Pleckstrin-homology.

The N-terminal portion of HOIP contains a PNGase/UBA or UBX (PUB) domain (Figure 1), which is reportedly an AAA-ATPase p97-interacting domain [24] that plays an important role to recruit linear ubiquitin-editing DUBs, such as OTULIN [25] and the CYLD-SPATA2 complex [26–29]. Thus, LUBAC forms complexes with negative regulators through the PUB domain. Furthermore, LUBAC includes several zinc finger domains (ZFs). Among them, the Npl4-type zinc finger (NZF) domain in HOIL-1L specifically binds linear ubiquitin [30], whereas the NZF domain in SHARPIN binds K63-ubiquitin to regulate the cell death pathway [31]. There are two NZF domains in HOIP, and NZF1 binds NEMO, a LUBAC substrate, during linear ubiquitination [32]. Collectively, these findings indicate that LUBAC consists of multiple functional domains for the regulation of linear ubiquitination and participates in various physiological phenomena.
