Regulation of cellular activities relies on various post-translational modifications (PTM), such as phosphorylation, glycosylation, or acetylation (1-3). Unlike the aforementioned PTM that transfers chemical moieties to the targets, ubiquitination ligates ubiquitin protein to the substrates (4, 5). The covalent attachment of the C-terminal carboxyl group of ubiquitin to the amine group of lysine residues on a target protein is called monoubiquitination. Ubiquitin also forms different types of chains, called polyubiquitin, through the ubiquitination on an amino group of 7 lysine residues (K6, K11, K27, K29, K33, K48, K63) or the first methionine (M1) residue of ubiquitin. The diverse conformational complexity of polyubiquitin chains gives cells abilities to differentiate and regulate various cellular processes. M1-linked linear polyubiquitin chain participates in inflammation and immune response. In the NF-kappaB (NF-kB) pathway, linear ubiquitin chain assembly complex (LUBAC) modifies NEMO, a subunit of inhibitor of kB (IkB) kinase (IKK) with M1 linkage that triggers phosphorylation of IkB (6-10). K6-linked polyubiquitin participates in DNA repair events and mitochondria stability (11-13), while K11- and K48-linked polyubiquitin is related to proteasomal degradation (14-17). K27-linked polyubiquitin controls DNA damage response and innate immune response. For instance, K27-linked polyubiquitination on histone 2A caused by RNF168 is related to DNA damage response (18). K29-linked polyubiquitination is associated with the Wnt signaling pathway, which regulates embryogenesis and tumorigenesis. K29-linked polyubiquitination on Axin, a scaffold protein in the Wnt signaling, disturbs interaction with LPR5/6 and inhibits the Wnt signaling (19, 20). It is known that K33 linkage participates in the regulation of T-cell antigen receptor and AMP-activated protein kinase-related protein kinase (21, 22). Together with K11-linked chain, K48 linkage plays a crucial role in the proteasome-dependent degradation pathway and ERAD pathway. Ubiquitin receptor on proteasome recognizes homotypic K48 linkage chain on the substrates and guides the substrate to enter the proteasome (15, 23). K63 linkage participates in proteasome DNA damage repair and endosomal-lysosomal system (24, 25). Also, K63 linkage plays an essential role in immune signaling. K63 polyubiquitin chain acts as docking sites in immune pathway proteins. K63-linked polyubiquitin chain on MALT1 is related to IkBα degradation and NF-kB activation (26). Moreover, K63 linkage is associated with immunomodulatory function in T cells and NLR-mediated signaling, and STING signaling in viral infections (27, 28).
The canonical ubiquitination system consists of three stages of the catalytic cascade (1, 29). The ubiquitin-activating enzyme (E1) hydrolyzes ATP and catalyzes acyl adenylation of the ubiquitin. The AMP-Ub intermediate forms a thioester bond with the catalytic cysteine of the E1 (1, 30). Next, Ub on the E1 is transferred to the catalytic cysteine on the ubiquitin-conjugating enzyme (E2) (31, 32). Finally, E2 works with the ubiquitin ligation enzyme (E3) to attach Ub to the substrate. E3 ligases are divided into three major classes (HECT, RING, and U-box, RBR). Each of them has a unique mechanism to transfer ubiquitin to a substrate (33, 34). Most E3 ligases make an isopeptide bond between the carboxyl terminus of the ubiquitin and the amino group of Lys on the target protein or preceding Ub (1). All E3 ligases bind to E2-Ub thioesters and catalyze the transfer of ubiquitin from E2 to the substrate lysine. In particular, HECT (Homologous to E6-associated protein C terminus) type E3s have catalytic cysteine that accepts ubiquitin from E2 via the transthioesterification reaction (35). In contrast, RING (Really Interesting New Gene) type E3 ligases have no active site residues and mediate direct ubiquitin transfer from E2 to the substrates. RING E3 ligase contains a RING (or RING-like) domain responsible for binding to E2 and stimulating the ubiquitin transfer (36). The RING domain generally adopts a cross-brace structure with two structural Zn2+ions. A related domain, known as a U-box, is similar in function and structural fold but has a hydrophobic core instead of the structural metal ion (37). The RBR (RING-between-RING) has two RING domains but combines roles of both RING and HECT E3 ligase (38, 39). One of the RING domains of RBR binds to E2 like the canonical RING domain, while the other RING domain accepts ubiquitin in a similar way to the HECT E3 ligases. Similar to other PTM, ubiquitination is also a reversible process, and the ubiquitin on the target proteins is recycled by deubiquitinase (DUB), which cleaves the isopeptide bond between ubiquitin and the substrate. About 100 types of DUBs have been identified in humans, and they are classified into seven superfamilies. Six of them (USP, OTU, MJD, UCH, MINDY, and ZUFSP) are cysteine proteases, whereas JAMM belongs to a zinc-containing metalloprotease (40, 41).
Some effectors have been demonstrated to interact directly with the host ubiquitination machinery using the U-box or F-box domain. Both LegU1 and LegAU13 are F-box proteins and integrate into mammalian SCF complexes (Skp, Cullin, F-box containing complex), and the LegU1 SCF complexes ubiquitinate host chaperone protein BAT3 (48, 49). Another Legionella effector, LubX (Lpg2830), is a U-box E3 ligase (50). Like the eukaryotic U-box, LubX has E2 specificity and interacts with eight E2s (UBE2D1, UBE2D3, UBE2D2, UBE2D4 UBE2E2, UBE2E3, UBE2W1, and UBE2L6) (51, 52). Intriguingly, LubX has two U-box motifs (U-box-1 and U-box-2). However, the LubX U-box-2 domain alone shows no E3 activity, whereas LubX U-box-1 is sufficient for interacting with E2s and catalyzes polyubiquitination. Structural analysis of LubX U-box-1 in a complex with human UBE2D2 reveals that most of the LubX U-box-1:UBE2D2 binding interface residues are not conserved in LubX U-box-2 (53). The overall structure of LubX:UBE2D2 is similar to the human UBE4B U-box:UBE2D3 complex (Fig. 1A) (54). However, residues at the binding interface differ from each other. Although both LubX U-box-1 and human UBE4B bind to the Ala-Pro-Ser hydrophobic patch on E2s, LubX U-box-1 makes additional hydrogen bond networks between Ile39-Arg5, Lys68-Ser91, and Arg75-Glu92. Importantly, these residues are not conserved in LubX U-Box-2. These structural analyses indicate that
Not only E3 ligases but also deubiquitinases are found in
The crystal structure of LotA OTU2, LotB, and LotC revealed that all three Lots consist of a catalytic domain (green) and an extended helical lobe (EHL) (yellow) (Fig. 1B). Because the EHL is not found in any other OTU family, EHL defines
RavD is another
SidE family effectors (Substrates of Icm/Dot transporter E, SidEs) are multi-domain proteins. They consist of a deubiquitinase domain, PDE (phosphodiesterase) domain, mART (mono ADP ribosyl transferase) domain, and a coiled-coil domain. When SidEs are translocated in the host cell, they catalyze a unique ubiquitination process on host proteins, called phosphoribosyl ubiquitination (Fig. 2A) (67). The mART domain on SidEs trans-fers the ADP-ribose moiety from NAD+ (nicotinamide adenine dinucleotide) to Arg42 of ubiquitin and generates ADP-ribosy-lated ubiquitin (ADPR-Ub). The ADPR-Ub is then processed by the PDE domain (68). PDE releases the AMP moiety from ADPR-Ub and produces phosphoribosyl ubiquitin (PR-Ub) in the absence of a substrate. When there is a substrate during the ADPR-Ub processing, PR-Ub is transferred to a serine residue on substrate proteins. Like canonical ubiquitination, deubiquitinases specific to PR-Ub are also present (46, 69). DupA and DupB (Deubiquitinase for PR-ubiquitination A and B) are also
MavC (More regions allowing vacuole colocalization C, Lpg2147) and MvcA (MavC paralog A, Lpg2148) were initially discovered as ubiquitin Gln40 deamidase (81). Structural and biochemical studies reveal that MavC binds to an activated Ube2N-Ub conjugate and catalyzes an intramolecular transglutaminase reaction to produce a covalent bond between Gln40 of Ub and Lys92 of Ube2N (82, 83). In contrast, MvcA cleaves ubiquitin from Ube2N and reverses the MavC-mediated Ube2N ubiquitination (Fig. 3A) (84). Another
This mini-review summarized current understandings of the
Interestingly, the non-canonical ubiquitination mechanisms—phosphoribosyl ubiquitination and transglutaminase-induced ubiquitination—are found only in
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2021R1C1C100396112 and 2018R1A6A1A0302560722) and the Yonsei University Research Fund of 2021 (2021-22-0050).
The authors have no conflicting interests.