Ubiquitination

2. Ubiquitination

Protein post-translational modifications (PTMs) widely contribute to the complexity and functional diversity of the proteome. These dynamic chemical modifications on proteins, which modulate the activity, localization, and ability to interact with other molecules, enable cells to swiftly response to environmental stimuli and delicately regulate intracellular signaling cascades. Ubiquitination is one of the key regulatory modifications among PTMs.

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2.1 Ubiquitin Machinery

Ubiquitin is a highly conserved 76-amino-acid-residue protein that ubiquitously exists in eukaryotic cells. Through a three-step sequential conjugation reaction, ubiquitin can be appended to the ε-amino group of a Lys within the substrate protein via its C-terminal Gly residue. Ubiquitin activating enzymes, the E1s, activate ubiquitin

through conjugating the C-terminal Gly of ubiquitin to their own Cys residue in an ATP-requiring manner. In the second step, activated ubiquitin is then transferred to catalytic Cys residue of the E2 ubiquitin conjugating enzymes, which could mediate the transfer of ubiquitin to substrate directly or indirectly. The last step is conducted by E3 ubiquitin ligases, which recognize specific protein substrates and catalyze the formation of a covalent amide isopeptide linkage between C-terminal Gly of ubiquitin and ε-amino group of a Lys within the substrate (Hershko and Ciechanover, 1998).

Different topologies of ubiquitination exist in cells which grant the system with versatility. Protein substrates can be modified with a single ubiquitin on a single Lys residue or multiple Lys residues, which is defined as monoubiquitination and multi-monoubiquitination respectively. As there are seven Lys residues and the first methionine residue within ubiquitin itself (K6, K11, K27, K29, K33, K48, K63, M1), each of them can also serve as an acceptor for subsequent ubiquitin moieties, leading to the formation of polyubiquitination chain. During the elongation of ubiquitination chains, different linkages between each ubiquitin moiety result in formation of homotypic or heterotypic chains with distinct conformations and functions in the cells.

Homotypic chains are constructed with homogenous linkages, through one of the seven Lys residues and N-terminal Met residue of ubiquitin. Heterotypic chains, on the other hand, can be classified into mixed chains, chains with alternating linkage types, or branched chains, in which more than one ubiquitin moieties extended from the preceding one (Komander et al., 2009a; Komander and Rape, 2012).

Proteins marked by ubiquitin were initially thought to be delivered and destructed by proteasome (Ciehanover et al., 1978; Wilkinson et al., 1980). After years of research, “canonical ubiquitin chains” are termed, which represent the Lys48-linked

chains and Lys63-linked chains that contribute to degradation of modified proteins through ubiquitin-proteasome system (UPS) and assembly of protein interaction complexes respectively (Chau et al., 1989; Deng et al., 2000). With the application of proteomic techniques like quantitative mass spectrometry, it was revealed that all ubiquitin linkages indeed coexist in cells (Peng et al., 2003; Xu et al., 2009). Apart from extensively studied Lys48-linked chains, Lys6, Lys11, Lys29-linked and branched chains are implicated to play roles in proteasomal degradation as well (Meyer and Rape, 2014). In addition, many non-proteolytic functions of ubiquitination, including regulating activity, localization, or interactions of ubiquitinated proteins, have also been reported, showing diverse functions of ubiquitin signals (Komander and Rape, 2012).

However, with only limited knowledge about atypical ubiquitin chains and heterotypic chains, biological functions and physiological roles of them still remain to be explored.

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2.2 Deubiquitinases (DUBs)

About 100 DUBs are predicted to exist in human genome. Based on the mechanism of catalysis, DUBs can be subdivided into two groups: the Cys proteases and the zinc-dependent metalloproteases. Among six DUB families, only the JAMM/

MPN+ DUBs are metalloproteases while members of USP, OTU, UCH, Josephine, MINDY are Cys proteases (Abdul Rehman et al., 2016; Komander et al., 2009a).

With only one-fifth of the quantity of E3 ligases, it is reasonable that the possible substrates of each DUB may surpass that of each E3 ligase. In accordance with the complexity and versatility of ubiquitin modification, DUBs must display numerous layers of specificity. It was revealed that many DUBs are able to distinguish between

different ubiquitin chain linkages, although the ability is not determined by family.

During the hydrolysis of ubiquitin chain, catalytic domains of DUBs interact with distal ubiquitin by its enzymatic S1 site and proximal ubiquitin by S1’ site. Given that the proximal ubiquitin contributes its Lys to the isopeptide bond, it is elicited that linkage specificity of DUBs is characterized by position and orientation of the proximal ubiquitin (Kulathu and Komander, 2012; Mevissen et al., 2013). Based on the concept, structure of enzymatic active site, accessory ubiquitin-binding domains (UBDs), specific sequences surrounding the ubiquitinated Lys on the proximal ubiquitin are thought to be involved in the determination of linkage specificity as well (Mevissen et al., 2013). The other layer of specificity lies to the recognition of substrates. Most of the DUBs contain additional protein-interaction domains that directly interact with the substrates or indirectly target them to a specific localization within the cells (Komander, 2010; Mevissen et al., 2013).

The general functions of DUB fall into three categories. First, DUBs are required for generating free cellular mono-ubiquitin since the precursors encoded by four human ubiquitin genes are produced into linear-fused poly-ubiquitins or ribosomal protein-fused ubiquitins. Second, DUBs remove the ubiquitin chains of the modified proteins to reverse the ubiquitin signals and to contribute to ubiquitin homeostasis through releasing reusable free ubiquitin. Third, DUBs may participate in the editing of ubiquitin chains by trimming the chains (Komander et al., 2009a).

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2.3 TRABID

TRABID, also named ZRANB1, is a member of the second largest DUB family - OTU family. Similar to most of the members in OTU family which display an intrinsic linkage specificity toward a particular subset of ubiquitin chain types, TRABID shows a preference in hydrolyzing Lys29- and Lys33-linked ubiquitin chains 40-fold more efficiently than Lys63-linked chains in vitro (Licchesi et al., 2012; Virdee et al., 2010).

The human TRABID protein contains 708 amino acids and consists of three N-terminal Npl4-like zinc finger domains (NZFs), two ankyrin repeat ubiquitin binding domains (AnkUBD), and a C-terminal A20-like OTU domain. Although the OTU domain of TRABID and A20 are closely related, the presence of evolutionarily conserved ankyrin repeats sequence upstream of TRABID’s OTU domain implies a different catalytic preference between TRABID and A20 (Komander and Barford, 2008; Licchesi et al., 2012). Consistent with this notion, the accessory ubiquitin binding domains, NZFs and AnkUBD, were reported to provide extra contributions to linkage specificity (Abdul Rehman et al., 2016; Komander et al., 2009b; Licchesi et al., 2012; Michel et al., 2015).

AnkUBD functions to enhance the specificity and activity of the enzyme. The S1 ubiquitin binding site within the OTU domain binds distal ubiquitin, whereas the AnkUBD interacts with hydrophobic Ile44 patch of proximal ubiquitin, serving as a S1’

ubiquitin binding site (Kulathu and Komander, 2012; Licchesi et al., 2012). Based on the general function of NZF domain, three N-terminal NZFs are thought to provide additional ubiquitin binding sites via binding to the hydrophobic Ile44 patch of ubiquitin (Hurley et al., 2006). The tandem NZFs were shown to recognize K63-linked or linear ubiquitin chains for their equivalent conformations (Komander et al., 2009b).

However, further studies implicate that NZF1 specifically interacts with K29/33-linked di-ubiquitin and captures K29-linked heterotypic polyubiquitin chains containing K48

linkages present in cells (Kristariyanto et al., 2015; Michel et al., 2015). Interestingly, catalytic inactive TRABID (C443S) but not wild-type TRABID has been reported to form highly dynamic puncta in cells depending on their NZF domains. Colocalization of mutant TRABID with K29 only, K33 only, K27 only and K63 only ubiquitin mutants also indicates the possibility that inactive TRABID might be trapped by its substrates bearing these atypical ubiquitin chains (Licchesi et al., 2012; Tran et al., 2008).

Unlike A20, the physiological role of TRABID has not been completely characterized. TRABID has been reported as a positive regulator of Drosophila and mammalian Wnt-β-catenin signaling pathway through cleavage of Lys63-linked ubiquitin chain on APC, resulting in stabilization of β-catenin, which then associates with TCF/LEF to facilitate transcription of Wnt target genes (Tran et al., 2008).

However, the other study that aimed to identify small molecule TRABID inhibitors failed to confirm the role of TRABID in Wnt signaling pathway, leaving this function controversial (Shi et al., 2012). Later, TRABID was found to be involved in Drosophila immune-deficiency (IMD) pathway by directly removing Lys-63 ubiquitin chain from dTAK1 thereby reducing immune signaling output and lifespan (Fernando et al., 2014).

More recently, TRABID was identified to function as an innate immunological regulator through reducing Lys29- and Lys11-linked ubiquitin chains on demethylase Jmjd2d.

Stabilized Jmjd2d mediates demethylation of histone H3, allowing transcription of genes encoding IL-12 and IL-23 to contribute to inflammatory T cells responses (Jin et al., 2016).

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