Protein ubiquitination is an essential cellular process that maintains protein homeostasis, regulates protein, and cell functions, and removes aggregated and misfolded protein. Disruption in function of any of the protein components of the ubiquitination pathway is associated with human diseases including cancers. An important member in the ubiquitination cascade is the very large E3 ligase family that directs substrate modification. The RING-type E3 ligases possess a cysteine/histidine-rich zinc-binding RING domain that confers ligase functionality. RING domains adopt a canonical ββα-fold. TRIM proteins represent a novel class of RING-type E3 ligase. TRIM proteins consist of an N-terminal RING domain followed by one or two B-box domains. The two types of B-box domains play essential roles in protein ubiquitination by contributing to substrate targeting, ligase activity enhancement, and redundancy of ligase activity. This review presents a general background of the B-box domains, a structural and functional comparison with RING domains, and a summary of recent work demonstrating their role in proteolysis. We discuss new findings that reveal B-box domains which are ubiquitous and are found in non-TRIM plant proteins without the adjacent RING domain, indicating that B-boxes are members of RING-class E3 ligases.
- E4 ligases
Protein ubiquitination is an essential cellular process that maintains protein homeostasis (proteostasis) and removes aggregated and misfolded protein that could recruit other proteins away from their normal cellular functions. It serves to regulate protein and cellular functions. Dysregulation of any of the protein component usually result in human diseases including cancers and birth defects. As such, the focus on protein ubiquitination has grown significantly in the past 2 decades, leading to extensive knowledge and new insights. A member of the ubiquitination cascade that has received considerable attention involves the large and growing family of E3 ligases. This family directs the last step in the reaction cascade by facilitating the ubiquitination of protein substrates. It is generally accepted that each E3 ligase has a specific or group of substrates (Figure 1).
There are several subgroups of E3 ligases, with the largest consisting of proteins with a RING domain that confer ligase functionality. The RING E3 ligase domains are cysteine- and histidine-rich sequences that bind two zinc ions in a unique cross-brace manner and adopt a canonical ββα-fold (Figure 1).
B-box and RING domains fall under the category of zinc-finger domains, which are present in a diverse family of proteins that includes transcription factors, ribonucleoproteins, proto-oncoproteins, and E3 ligases . Zinc-finger domains or proteins are characterized as having cysteine and histidine residues arranged in one of several motifs that are relatively conserved in other proteins . The thiol group (S−) of the cysteine and a nitrogen atom of the histidine imidazole side-chain tetrahedrally bind a zinc ion .
Most zinc-finger proteins typically coordinate either a single zinc ion or two zinc ions, depending on the number of cysteine and histidine residues and their position within the sequence . A defining property of zinc-finger domains is that zinc coordination is required to stabilize the tertiary structure. Loss of zinc coordination by a mutation of any of its cysteine or histidine residue results in complete unfolding of the protein structure. Protonation of the cysteine or histidine by decreasing the pH of the protein solution will also result in unfolding. For domains that bind two zinc ions, disruption of coordination of one zinc ion is usually accompanied by the loss of binding of the other zinc ion, causing the domain to become unfolded rather than partially folded with one zinc ion [4, 5].
Zinc-finger domains were identified in the mid-1980s within the
For the next 2 decades, the number of proteins observed with the RING and B-box domain pairs would have increased, with most belonging to TRIM proteins that are defined by their N-terminal RING, B-box, and coiled-coil (RBCC) domains [11, 12]. This RBCC domain arrangement is conserved and found in all multicellular organisms . In humans, the RBCC domain is observed in a family of over 50 proteins; although few have been characterized in detail, their importance is underscored by the fact that some are oncoproteins (e.g., PML, RFP, and TIF1a), while others, when mutated, give rise to various congenital abnormalities [13, 14]. Members of this large protein family are found to play regulatory roles in a variety of cellular processes, including sperm vesicle exocytosis and intracellular release of HIV [15, 16]. The RBCC domain arrangement indicates at the very least that TRIM proteins have an overall common function. The RBCC proteins can have quite diverse C-terminal domain arrangements .
Interestingly, many TRIM proteins possess three consecutive cysteine/histidine-rich regions, the first being the RING domain. The other two domains are referred to as B-box1 and B-box2 domains (). While the nomenclatures suggest that the two types of B-box domains are homologous, they do not share any discernable sequence similarity with each other or with RING domains. A single B-box domain in TRIM proteins is always of the type 2 form (B-box2), while TRIMs with two have the B-box1 domain preceding the B-box2 domain. The name may have persisted to prevent confusion in distinguishing the presence of the two types of B-box domains in TRIM proteins. The B-box1 domain is slightly larger (50–60 aa) with a zinc-binding consensus sequence of C-X2-C-X7–12C-X2-C-X4-C-X2-[C/H]-X3–4-H-X4–9-H
3. Description of B-box domain structures
Despite their prevalence and location downstream of RING domains in TRIM proteins, very little was initially done to characterize the structures and functions of B-box domains. We postulate that this might have been so because of difficulties in obtaining sufficient quantities of the B-box domains for structural and functional studies. Indeed, each type of the B-box domain has proven to be quite challenging to express and purify using
The first comprehensive structural studies of B-box domains were based on the TRIM18/MID1 protein [13, 21]. Human MID1 is required for proper midline development during embryogenesis ([22, 23, 24, 25, 26]). Mutations of MID1, some of which are found within the B-box domains, are associated with X-Linked Opitz G/BBB syndrome (XLOS), a congenital disorder characterized by clefts of the lip and palate, cardiac structural defects, and genital anomalies [14, 27].
The structure of the B-box1 domain (residues Gln87-Pro165) was solved in 2006 by analyzing multidimensional data acquired by nuclear magnetic resonance (NMR) spectroscopy. The B-box1 domain was observed to coordinate two zinc ions in a cross-brace manner with six cysteine and two histidine residues (Figure 1) . Residues Ala115 to Pro165 form the core of the structure, while the preceding 30 amino acids are unstructured and initially included to aid in solubility. The structure consists of a two-turn α-helix that is preceded by a long structured loop consisting of two short β-strands separated by a type-2 β-turn. Two cysteine residues within the first part of the structured lasso-like loop1 coordinate one zinc ion with two other cysteine residues located within the first helical turn of the helix. Two cysteine residues that are part of the β-turn and two histidine residues, one located at the end of the α-helix and the other on the loop2 that follows the helix, coordinate the second zinc ion. The overall structure is very similar to the ββα-canonical RING fold (Figure 1).
The structure of MID1 B-box2 domain was solved a year later, using NMR data. In contrast to the MID1 B-box1 domain, the MID1 B-box2 consists of seven classical cysteine and histidine zinc-binding residues, suggesting that only one zinc might be coordinated by four of these residues (Figure 1). Sequence alignment of TRIM B-box2 domains reveals that approximately half of B-box2 domains consist of aspartate residues and the other half a cysteine residues in the same location. This observation suggests that Asp must be a highly conserved change  that should be performing the same role as the cysteine residue. Indeed, the MID1 B-box2 domain coordinates two zinc ions in a similar cross-brace manner as the B-box1 and RING domains. Two histidine, cysteine, and aspartate residues coordinate one zinc ion. The carboxylate oxygen of this conserved aspartate side chain participates in zinc coordination. The aspartate residue forms the necessary zinc-knuckle conformation with a cysteine residue two positions away (CxxD) to tetrahedrally coordinate the zinc ion [2, 21]. Although carboxylate groups are involved in binding catalytic zinc ions, for example, carbonic anhydrase [28, 29, 30], or other non-structural metals, this was the first demonstration in a zinc-finger protein. The B-box2 domain adopts a two-turn α-helix, two short β-strands separated by a type-2 β-turn, and two structured loops adjacent to the helix. Despite a lack of sequence similarity, the structures of the two types of B-box domains are remarkably similar (Figure 1C). The positions of the two zinc ions are in similar locations, namely near the N-terminus of the helix and to the bottom left of the helix (given the specific orientation shown in Figure 1B). Importantly, the mechanism of zinc coordination (cross-brace) and the ββα-fold are comparable to those of RING domains.
Structures of the B-box1 domain from TRIM19 and the B-box2 domain from TRIM1/MID2, TRIM5α, TRIM21, TRIM29, TRIM39, TRIM41, TRIM54, and TRIM63/MuRF1 have been solved. All the B-box domain structures are similar. Consequently, we conclude that MID1 consists of three consecutive domains with RING folds. Thus, the TRIM protein family must represent a new class of E3 RING-type ligase, consisting of two or three consecutive RING folds.
To identify a possible role of the two adjacent B-box domains, the structure of both was determined in their native tandem form (res A110-E214). The two B-box domains maintained their original structures and pack against each other with the interface formed by residues located on the structured loop-1 near the two antiparallel β-strands. The surface area of the interface is 188 Å2 (17% of the total surface). Interestingly, the tandem globular structure is very reminiscent of the intermolecular association observed for heterodimeric RING structures, such as the BARD1 and BRCA1 domains (12) and the polycomb group protein (Bmi-1) and Ring1B polycomb group (14), and the homodimeric RINGs, such as HDM2 , RNF4 and 8 [32, 33], and cIAP2 . The TRIM19 B-box1 and TRIM54 B-box2 domains were solved as symmetric dimers by X-ray crystallography. The structures of RING dimers reveal the domains interacting via residues located on and near loop-1. The BRCA1-BARD and RNF8 dimers also include adjacent structures, such as helical dimers. The area of the interface of the hetero- and homo-RING dimers is approximately 150–200 Å2, similar to that observed for the MID1 B-box1,2 heterodimer. In spite of their interactions, it appears that in the case of MID1, unfolding of the B-box1 structure, via a mutation of one its zinc-binding residues, had little effects on the structure and stability of the B-box2 domain . This observation suggests the possibility that each B-box domain could function independently or have redundant E3 ligase function.
The structures of two B-box domains were solved in complex with another TRIM domain. The TRIM5α B-box2 was crystallized with its coiled-coil domain, which contributes to oligomerization. Binding studies using NMR and dynamic light scattering using a TRIM5α proteins with a native and mutant B-box2 domain reveal that the B-box2 domain contribute to higher order self-association . Given that the B-box2 domain is required for substrate ubiquitination, self-association may contribute to enhanced E3 ligase activity and substrate targeting [36, 37] of the native TRIM5α. The B-box domains of TRIM27 are also determined to be crucial for multimerization by possibly helping to orient the coiled-coil domain in a way that maintained the multimer interaction . In contrast, the B-box2 domain of TRIM21 was crystallized with the N-terminal RING domain, and the structure reveals that the B-box2 domain interacts with the RING domain on a surface that is important for RING-E2 interaction. In this case, the structure suggests that TRIM21 B-box2 may have an autoinhibitory effect, although further studies are required. It is possible that the structures of these complexes may be affected by protein packing within the crystal lattice. More work needs to be done to understand the mechanism of function of these B-box domains, which we postulate are now key players in the ubiquitination field.
4. A brief description of ubiquitination and the role of E3 ligases
In order to appreciate the function of RING and B-box domains, a brief summary of protein ubiquitination is provided. All living organisms employ a fairly common mechanism to recycle proteins so as to regulate protein function, proteostasis, and cell cycle. Eukaryotic cells employ protein ubiquitination, a post-translational modification using the highly stable 76-amino acid ubiquitin protein (Ub) [39, 40, 41]. Bacteria use prokaryotic ubiquitin-like protein (Pup) in an analogous manner . Polyubiquitinated proteins, usually with a chain of at least four-linked Ub, are targeted to the proteasome where they are proteolytically cleaved into peptides [39, 40, 43]. Ubiquitin chains can form via any of its seven lysine residues or combinations of the seven; homogenous chain links, example K48, promote protein degradation [44, 45, 46], but some have signaling functions [47, 48]. The Ubs are cleaved by deubiquitinating enzymes (DUBS) and recycled . Although mono- and diubiquitinated proteins are directed to the proteasome , there is evidence that this level of modification serves a signaling role, in which modified proteins can have their functions and cellular location altered. The monoubiquitination of cytosolic proteins results in translocation to the nucleus to participate in DNA repair, transcription regulation, and inflammatory response [39, 51, 52, 53, 54].
Ubiquitination involves three classes of enzymes. First, the E1-activating enzyme (E1) catalyzes the adenylation of the C-terminal glycine of Ub (Ub~AMP). This phosphoester bond undergoes a nucleophilic attack by the sulfhydryl group of the active site cysteine residue on the E1. In the next step, the Ub is transferred to an active site cysteine residue on a family of E2 conjugating enzymes (E2) to form an activated thioester-linked E2~Ub complex [39, 43]. Typically most types of E2 enzymes require the concerted action of an E3 ligase (E3) to target and facilitate substrate ubiquitination [40, 43]. There are several classes of E3 ligases: the homologous to the E6-AP Carboxyl Terminus (HECT), RING-InBetweenRing-RING families, and RING class [39, 40, 41, 50, 55]. The HECT and RING-IBR-RING families accept the Ub via a trans-thiolation reaction from the E2 before transferring it to the target protein. The RING E3 ligase, which includes the Skp-Cullin-F-box (SCF) complex, U-box, and now the B-box, represents the overwhelming majority of E3 ligases. While the mechanism is unclear, the RING-type ligase binds and places in close proximity to the target protein and the E2 enzyme. With the SCF complex, the RING domain (aka RBX) recruits the E2~Ub, while another SCF subunit (usually, the F-box) binds the substrate. In the last reaction, the E2~Ub thioester bond undergoes a nucleophilic attack (thiolysis) by a lysine residue of the target protein whereby the side-chain amino group forms an isopeptide bond with the C-terminal carboxylate group of Ub. Subsequent Ubs can be attached to other lysine residues of the substrate, but more commonly observed to form a polyubiquitin chain with linkages to one of seven lysine residues of the Ub [39, 40, 43]. Chains can be formed with Lys 6, 11, 27, 29, 33 and the N-terminus (M1) amino group, but the two more common reported linkages involve Lys48 and Lys63 . We argue that the level or amount of polyubiquitination or Ub processivity can be an assessment of the level of E3 ligase activity by a RING protein.
Typically, confirmation that a RING domain/protein possesses E3 ligase activity is accomplished by performing
5. Possible mechanism of action of RING E3 ligases
As noted, the mechanism of function of RING E3 ligases is unclear, but considerable progress has been made to provide insights. The structures of several E2-RING complexes reveal that the RING domain is positioned ~15 Å from the active site and the thioester linkage between the E2 and Ub [59, 60]. Based on these structures, it is unclear how the RING domain affects reactivity or electrophilicity on the E2~Ub linkage. To gain insights on the role of RING E3 ligases, Klevit and co-workers [61, 62] used molecular dynamic and NMR studies to show that the bound RING E3 ligase promoted a “closed” E2~Ub conformation, whereby the Ub populates one interaction mode with slightly greater frequency. In the absence of a bound RING domain, the covalently attached Ub is highly mobile and does not favor any specific surface of E2 to interact . There is no fixed or stable structure between the Ub and E2 proteins. Promoting the positioning of the Ub to the “back” surface of the E2 reduces steric hindrance for nucleophilic attack by the incoming lysine residue. In addition, key amino acid interactions at the E2-RING binding interface appear to contribute to the activation of the thioester bond. For example, residue Gln92 of UbcH5 (Ube2D1), which is located on a helical turn adjacent to active site Cys-85, forms a hydrogen bond with an arginine or lysine residue on loop-2 of the RING domain. Disruption of this interaction through mutation of Gln92 or the arginine severely disrupts the rate of Ub transfer [62, 63]. In fact, the rate of thiolysis with free lysine, as the substrate, for either mutant is comparable to that of E2~Ub without a bound RING domain. Thus, it is generally accepted, given what is known from the various E2-RING interaction studies that the RING domain contributes allosterically to electrophilicity of the thioester bond.
6. The B-box domains are new members of the RING E3 ligase
Given that the B-box domains have similar RING folds, it was postulated to function similarly. First confirmation that the B-box domains possess E3 ligase activity was demonstrated with MID1 . The
Intriguingly, the tandem B-box domains also exhibit weak E3 ligase activity, with no greater level of autoubiquitination activity than that observed with the B-box1 domain . This is in contrast to hetero- and homodimeric RING dimers, which exhibited greater activity than the mono form [32, 33, 65]. The BRCA1-BARD1 complex, in which BRCA1 (breast cancer 1) heterodimerizes with BARD1 (BRCA1-associated RING domain), exhibited enhanced activities compared to BRCA1 alone; BARD1 does not exhibit ligase activity [65, 66, 67, 68]. Enhancements of activities were also observed for MDM2/HDX , RNF4 , inhibitor of apoptosis (IAP) proteins , BMI1-RING1 , and membrane-associated RING-CH family of E3 ubiquitin ligases (MARCH1) RING dimers , to name a few. The mechanism of E3 ligase enhancement by RING dimers or the lack of enhancement by the MID1 B-box domains is unclear. However, there are several publications that proposed rationales of the role of RING dimers [32, 73], but they will not be discussed here.
Despite the MID1 B-box domains not showing strong ligase activity, studies with TRIM16 revealed that its B-box domains exhibited greater level of activities. There were substantial amount of polyubiquitinated products, as demonstrated by the intensity of the smearing observed by Western blot analysis . Both
7. Tandem RING and B-box domains are more active: could it be E4 ligases?
To understand the role of the B-box domains in the context of being adjacent to the RING domains, as they are commonly found in TRIM proteins, autoubiquitination assays were performed with the MID1 RING domain in tandem with B-box1 (RING-B-box1 (RB1)) and both B-box domains (RING–B-box1–B-box2 (RB1B2)). The goal was to determine whether each domain functions independently or if they have synergistic contribution to justify a possible evolutional reason for their presence in tandem. In the case of RB1, greater amount of polyubiquitinated products were observed compared with the results of the ubiquitination assay with the RING domain alone [14, 64, 76]. The rate of product formation was qualitatively faster. Whereas polyubiquitinated products were observed with the MID1 RING domain after 120 minutes, polyubiquitinated products were observed within the first 5–10 minutes of the assay with RB1. Similarly, the MID1 RB1B2 protein construct exhibited comparably rapid ligase activity as the RB1 domain construct. Within experimental error, it was difficult to determine whether there was greater or lesser amount of polyubiquitinated products. Therefore, it was difficult to identify the contribution of the B-box2 domain within the RB1B2 construct.
To probe whether the B-box2 domain contributes to ligase activity as part of the RB1B2 construct, a C142S mutation was introduced within the B-box1 domain (RB1*B2). Cysteine-142 coordinates one of the two zinc ions, and its mutations to serine resulted in the loss of coordination of both zinc ions and unfolding of the B-box1 domain . By Western blot, the E3 ligase activity of the RB1*B2 protein construct was indistinguishable from the RB1B2 construct, indicating that the B-box2 domain can compensate for the loss of function of the B-box1 domain. To confirm that the B-box2 domain has the same enhancing role as the B-box1 domain, an RB1* protein construct was designed, and the activity was observed to be similar to that of just the RING domain .
These observations indicate that the B-box domains, by some unknown mechanism, appear to enhance the E3 ligase functionality of the adjacent RING domain. It is wholly possible that the enhancement observed could be that both RING and the B-box domains have gained E3 ligase activities, there is some synergy in activities, or that the B-box domains may function as E4 ligases [77, 78, 79, 80]. E4 ligases are domains with a RING fold that enhance the ligase activity of RING E3 ligases. Possible examples of E4-enhancing ligases are the BARD1 and HDMX RING domains. In the mid-2000s, the U-box domain, which adopts a similar ββα-RING fold but without the coordination of zinc ions (), was initially shown to play an E4-enhancing role for RING E3 domains . Subsequently, it was concluded that U-box domains can function as E3 ligases and now represent a new member of the RING-type E3 ligases with a similar mechanism of action as RING domains [61, 83, 84].
The function of the B-box domains of TRIM5α, TRIM25, and TRIM32 is also studied [85, 86]. TRIM5α possesses anti-viral/anti-HIV activities [56, 87]. TRIM25 plays a crucial anti-viral role by ubiquitinating the N-terminal caspase activation and recruitment domains (CARDs) of the recognition receptor RIG-I [86, 88]. Mutations of TRIM32 are associated with limb-girdle muscular dystrophy type 2H. TRIM25 consists of RING, B-box1, and B-box2, while TRIM5α and TRIM32 have the RING and B-box2 domains. TRIM25 possesses both Ub and interferon-stimulated gene 15 (ISG15)-E3 ligase activities [86, 88]. ISGylation serves more of a signaling role, as ISG15-modified proteins have altered functions . For these proteins, the B-box domains are required for enhanced activities. Using thiolysis assays (nucleophilic attack on the thioester of charged E2~Ub by lysine), the role of the B-box domains was assessed for TRIM25 and 32 . The constructs with the RING domain alone marginally activated the reaction, but those including the B-box domains significantly accelerated thiolysis.
In the case of TRIM5α, autoubiquitination assays with monomeric RING alone did not produce polyubiquitinated products/chains. In contrast, the RB2 and RB2CC protein constructs showed considerable increases in activities, which were attributed to the presence of the B-box domain. As control, the RB2*CC protein construct with a destabilizing B-box2 mutant resulted similar levels of ubiquitination products as just with the RING domain .
In summary, the results from the RB1, RB1B2, RB1*B2, and RB1* autoubiquitination assays of MID1/TRIM18, TRIM25, and TRIM32 suggest that TRIM proteins with RING and two B-box domains have some redundancies in the enhancement role of the B-box domains. Furthermore, given that the RING-less TRIM16 tandem B-box possesses strong ligase activity adds to the support that the B-box domains can possibly have dual roles, functioning as E3 and E4 ligases.
8. The B-box domains are required for substrate polyubiquitination
For all three substrates, full-length MID1 was shown to catalyze their polyubiquitination [14, 76, 91]. The role of the B-box domains for substrate ubiquitination was demonstrated for PP2Ac and alpha4 [14, 76]. With just the RING domain, a weak band was observed on the Western blot, indicating low amount of mono-ubiquitinated products [14, 76]. In contrast, polyubiquitinated products were observed with the RB1 and RB1B2 protein constructs. The results of the assays with the RB1* (C141S) protein construct yielded monoubiquitinated PP2Ac and alpha4 products, confirming the B-box1 domain is important for substrate targeting and polyubiquitination. The RB1*B2 protein construct catalyzed the polyubiquitination of PP2Ac but not alpha4. These results indicate a few things: the B-box binding of protein substrates is a critical role for polyubiquitination, the B-box2 domain can compensate for the unfolded B-box1 domain, and the B-box2 domain can contribute to some B-box1 redundancies in MID1’s overall E3 ligase activity. The levels of ubiquitination of PP2Ac and alpha4 parallel the results observed with the autoubiquitination assays, confirming the various roles of the B-box domains. Similar results were observed with TRIM5α TRIM25, TRIM19/PML, and TRIM63.
TRIM5α targets the HIV capsid protein. Deletion of the B-box2 domain affected oligomerization state, E3 ligase activity, and substrate ubiquitination [35, 37, 56]. TRIM25 targets RIG-I.
Finally and importantly, studies with TRIM27/rfp revealed a central role of the B-box domain in substrate binding and ubiquitination and subsequent degradation . This report was the first demonstration that E3 ligase activity of TRIM27, a TRIM protein with a RING domain, is conferred to the B-box domain instead . This unique finding has not, to our knowledge, been observed with any other TRIM proteins containing both RING and B-box domains.
9. MID1 B-box1 domain E3 ligase activity can be enhanced
As noted, we postulate that RING E3 ligases that exhibit different levels of
Intriguingly, even though the P151L mutant B-box1 possesses greater activity, substrate ubiquitination assays revealed that the mutation disrupts binding and targeting of the alpha4 protein. This observation strongly supports our hypothesis that RING E3 ligase activities may be a compromise between level of activity and substrate binding, as defined evolutionarily. In unpublished work, we have identified several specific amino acids in RING domains that are important for RING–E2 interaction but that are not present in B-box1 domains. Introduction of these amino acids into the MID1 B-box1 domain resulted in significant increases in auto-ubiquitination activity, including polyubiquitination (unpublished).
10. Structural comparison of RING and B-box domains
In light of our findings that the E3 ligase activity of the MID1 B-box1 domain can be enhanced, we examine the structures of RING and B-box domains to understand if there may be additional features that can rationalize the difference in activities of MID1 RING and B-box domains. While it is not feasible to provide detailed analyses of all the differences between the various RING and B-box domain structures, we make general qualitative comparisons. As noted, the overall structures of RING and B-box domains are similar (Figure 1). In the case of the MID1 B-box domain, the position and size of loop2 may contribute to their decreased E3 ligase activities (Figure 4A). The sequences of a few RING and B-box domain are aligned, and distributions of amino acid types on the E2-binding surface on the RING and B-box domains are depicted for comparison (Figures 3 and 4). There are some key differences in amino acids between the RING and B-box domains that may also contribute to differences in the level of activity. However, those will not be discussed in detail here. The exact mechanism of E2-Bbox binding has not been characterized, and therefore, for the following discussion, we make the assumption that the B-box domains interact with the E2 enzyme in a similar manner as RING domains. The different types of residues (hydrophobic [green], acidic [red], basic [blue], and uncharged polar [gray]) are displayed. We used the HDM2 and human Fanconi anemia (fancl) RING domains [31, 120]. On the E2-binding surfaces, both RING domains (Figures 3 and 4) show predominantly hydrophobic residues. With the structure of the HDM2 RING domain (PDB 2hdp), there is also a large adjacent basic patch on the outer surface of the helix, but its role in E2 binding is not clear. There is also a basic residue on loop2 and a small acidic patch toward the top of the structure, and these participate in Ube2D2 E2 binding. For the fancl RING domain, the large hydrophobic patch is located in same region, but there are no large charged surfaces. Evaluation of fancl-RING-Ube2T complex (PDB 4ccg) reveals that the E2 enzyme interacts predominantly with the hydrophobic region of the RING domain (Figure 4B) . Structures of other RING-E2 complexes reveal similar types of interactions.
In contrast, the hydrophobic patches on the MID1 B-box1 and B-box2 structures are smaller and not as contiguous as those observed with the HDM2 and Fancl RING structures (Figure 3). There are more charged residues on the surface. The PML/TRIM19 B-box1 domain (PDB 2mvw)  has more polar residues distributed instead of hydrophobic residues. Two smaller hydrophobic patches are observed on opposite sides. The distribution of residues for the TRIM5α B-box2 domain (PDB 2ecv) is very similar to that of the MID1 B-box1 structure. These comparisons reveal that there are differences in amino acid types at the canonical E2-binding site that might influence the mechanism of interactions between RING and B-box domains with their cognate E2 enzymes and hence the level of activity.
Interestingly, the structure of fancl RING domain with Ube2T E2 reveals that Ube2T does not have a corresponding Gln92 residue to form a hydrogen bond with a basic residue on loop2 of the RING domain, which is present is several RING and U-box domains. Instead, the complementary positions consist of hydrophobic residues, suggesting that allosteric effects of RING binding might be transmitted via hydrophobic interactions. In contrast, the HDM2 RING domain has an arginine that can form a hydrogen bond with Gln92 of the Ube2D2/UbcH5 E2 enzyme. This interaction is important for allosteric effects to influence cleavage of the thioester bond. It is possible that these subtle differences in binding mechanisms might provide a rationale for differences in the level of E3 ligase activities observed for RING-type E3 ligases. Differences in activity may also be due to mismatch in cognate E2-RING partners with
11. B-box2 domain may additionally possess a regulatory role
In addition to the above noted roles of the B-box domains, it has been suggested that the presence of MID1 B-box2 domain impacts the binding efficiency of the B-box1 domain to alpha4. Binding studies with the MID1 and alpha4 proteins revealed tightest binding with a RB1 construct and reduction in binding with RB1B2 and larger MID1 constructs . The apparent reduction in RB1-alpha4 binding may due to the B-box2 domain binding in an overlapping site with alpha4 . Interestingly, we saw a reduction of the band intensities of the polyubiquitinated products of both auto- and alpha4-ubiquitinations with RB1B2 , indicating that B-box2 domain is regulating the alpha4 interaction. On the other hand, it is possible that the decrease is due to the two B-box domains affecting the RB1B2 interaction with the E2 enzyme.
Another TRIM protein for which the B-box2 domain has been ascribed a possible regulatory function is TRIM21, which is involved in immune signaling and is found in almost all cell types and tissues in mammals. In contrast to MID1, the TRIM21 RING domain exhibited greater ligase activity than the RB2 protein construct . The result is confirmed by the E2~Ub thiolysis assays: the rate of Ub discharge was greater with the RING than with the RB2 domain construct. NMR experiments confirmed that the RING and B-box2 domains interact via the surface important for self-oligomerization .
12. The role of B-box domains in RINGless-TRIM proteins
There are currently six characterized human TRIM proteins that lack the N-terminal RING domain: TRIM14, TRIM16 (EBBP), TRIM20 (PYRIN/MEFV), TRIM29 (ATDC), TRIM44 (DIPB), and TRIM66. RINGless TRIM proteins are found in
TRIM16 is a transcriptional regulator involved in regulating neuroblastoma cell growth, migration, and tumorigenicity . Apparently, it can function as E3 ligase via both homodimerization and heterodimerization with TRIM18, TRIM19, and TRIM24 . The tandem B-box domains are capable of very weak homodimerizing interactions in the absence of the coiled-coil domain.
Very little is known about the other RINGless-TRIM proteins (Figures 5 and 6). TRIM29 is an oncogene that regulates p53 and is overexpressed in many different cancers including breast, lung, bladder, and pancreatic [128, 129]. It can form both homodimers and heterodimers with TRIM1/MID2, TRIM11, TRIM23, and TRIM27 . Despite the lack of the RING domain,
While the mechanism of action is not clear, it is possible that RINGless-TRIM proteins function through homo- or heterodimerization via at least one of its B-box domains. Self-association/oligomerization through the B-box domains is shown to contribute to E3 ligase activity possible via an apparent localized concentration effect.
13. B-box domains are found in plants
While the majority of B-box domains are found in mammals, recent publications have identified 32 B-box proteins (known as BBX proteins) in
Defining specific functions to the B-box domains of BBX proteins is lacking, probably because of their recent realization in plants. Without specifics, a large number of these BBX proteins are postulated to be involved in the ubiquitination pathway. Several BBX proteins are shown to interact with an
While it is clear that there is still much to be learned about TRIM proteins and their E3 ligase activity, there has been a great push toward better understanding of the role of the B-box domains over the past decade. Although the RING domain has received much of the spotlight, it is now clear that the B-box domains are integral for substrate binding/targeting, protein ubiquitination, and enhancement or activation of the ligase activity of TRIM and BBX protein families. B-box domains have diverse roles that include protein-protein interactions, substrate ubiquitination and sumoylation, and transcriptional regulation. What is not clear is whether B-box domain really served as E4-enhancing ligase to enhance the ligase activity of the RING domains, as observed in dimer RING E3 ligases, or whether they synergistically gain activity alongside the RING domains. Their role in contributing to oligomerization for some TRIM and BBX proteins to account for enhanced E3 ligase activity may be due to an apparent increase in the localized concentration of the TRIM or BBX protein. However, more studies are needed. Progress will require multifaceted approaches involving structure determination, protein-protein binding studies, and functional assays. Nonetheless, we hope that sufficient evidence have been provided, including those of TRIM18/MID1 and TRIM27, demonstrating that B-box domains are and should be considered E3 ligases and not as a supporting player to RING domains.
The author thanks Dr. Katharine Wright for her work in the lab and for contributing ideas for this review, and Ms. Jessica Webb for her help and contributions with the figures. This work was supported in part by a National Science Foundation grant (MAM, 1808391).
Klug A. The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation. Quarterly Reviews of Biophysics. 2010; 43:1-21
Massiah MA, Blake PR, Summers MF. Nucleic Acid Interactive Protein Domains that Require Zinc. New York, NY: Oxford University Press; 1998
Berg JM. Zinc fingers and other metal-binding domains. Elements for interactions between macromolecules. The Journal of Biological Chemistry. 1990; 265:6513-6516
Wright KM, Wu K, Babatunde O, Du H, Massiah MA. XLOS-observed mutations of MID1 Bbox1 domain cause domain unfolding. PLoS One. 2014; 9:e107537
Zhao Y, Zeng C, Massiah MA. Molecular dynamics simulation reveals insights into the mechanism of unfolding by the A130T/V mutations within the MID1 zinc-binding Bbox1 domain. PLoS One. 2015; 10:e0124377
Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. The EMBO Journal. 1985; 4:1609-1614
Neuhaus D, Nakaseko Y, Nagai K, Klug A. Sequence-specific [1H]NMR resonance assignments and secondary structure identification for 1- and 2-zinc finger constructs from SW15. A hydrophobic core involving four invariant residues. FEBS Letters. 1990; 262:179-184
Churchill ME, Tullius TD, Klug A. Mode of interaction of the zinc finger protein TFIIIA with a 5S RNA gene of Xenopus. Proceedings of the National Academy of Sciences of the United States of America. 1990; 87:5528-5532
Reddy BA, Etkin LD. A unique bipartite cysteine-histidine motif defines a subfamily of potential zinc-finger proteins. Nucleic Acids Research. 1991; 19:6330
Lovering R, Hanson IM, Borden KL, Martin S, O'Reilly NJ, Evan GI, et al. Identification and preliminary characterization of a protein motif related to the zinc finger. Proceedings of the National Academy of Sciences of the United States of America. 1993; 90:2112-2116
Ogawa S, Goto W, Orimo A, Hosoi T, Ouchi Y, Muramatsu M, et al. Molecular cloning of a novel RING finger-B box-coiled coil (RBCC) protein, terf, expressed in the testis. Biochemical and Biophysical Research Communications. 1998; 251:515-519
Short KM, Cox TC. Sub-classification of the rbcc/trim superfamily reveals a novel motif necessary for microtubule binding. The Journal of Biological Chemistry. 2006; 281(13):8970-8980
Massiah MA, Simmons BN, Short KM, Cox TC. Solution structure of the RBCC/TRIM B-box1 domain of human MID1: B-box with a RING. Journal of Molecular Biology. 2006; 358:532-545
Du H, Wu K, Didoronkute A, Levy MV, Todi N, Shchelokova A, et al. MID1 catalyzes the ubiquitination of protein phosphatase 2A and mutations within its Bbox1 domain disrupt polyubiquitination of alpha4 but not of PP2Ac. PLoS One. 2014; 9:e107428
Uchil PD, Quinlan BD, Chan WT, Luna JM, Mothes W. TRIM E3 ligases interfere with early and late stages of the retroviral life cycle. PLoS Pathogens. 2008; 4:e16
Kitamura K, Tanaka H, Nishimune Y. Haprin, a novel haploid germ cell-specific RING finger protein involved in the acrosome reaction. The Journal of Biological Chemistry. 2003; 278:44417-44423
Short KM, Cox TC. Subclassification of the RBCC/TRIM superfamily reveals a novel motif necessary for microtubule binding. The Journal of Biological Chemistry. 2006; 281:8970-8980
Klug A, Schwabe JW. Protein motifs 5. Zinc fingers. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology. 1995; 9:597-604
Massiah MA, Wright KM, Du H. Obtaining soluble folded proteins from inclusion bodies using Sarkosyl, triton X-100, and CHAPS: Application to LB and M9 minimal media. Current Protocols in Protein Science. 2016; 84(6):13.1-6-13.1-24
Tao H, Liu W, Simmons BN, Harris HK, Cox TC, Massiah MA. Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS. Biotechnology Techniques. 2010; 48:61-64
Massiah MA, Matts JA, Short KM, Simmons BN, Singireddy S, Yi Z, et al. Solution structure of the MID1 B-box2 CHC(D/C)C(2)H(2) zinc-binding domain: Insights into an evolutionarily conserved RING fold. Journal of Molecular Biology. 2007; 369:1-10
Cox T. Taking it to the max: The genetic and developmental mechanisms coordinating midfacial morphogenesis and dysmorphology. Clinical Genetics. 2004; 65:163-176
Cox TC, Allen LR, Cox LL, Hopwood B, Goodwin B, Haan E, et al. New mutations in MID1 provide support for loss of function as the cause of X-linked Opitz syndrome. Human Molecular Genetics. 2000; 9:2553-2562
Granata A, Quaderi NA. The Opitz syndrome gene MID1 is essential for establishing asymmetric gene expression in Hensen's node. Developmental Biology. 2003; 258:397-405
Quaderi NA, Schweiger S, Gaudenz K, Franco B, Rugarli EI, Berger W, et al. Opitz G/BBB syndrome, a defect of midline development, is due to mutations in a new RING finger gene on Xp22. Nature Genetics. 1997; 17:285-291
Schweiger S, Schneider R. The MID1/PP2A complex: A key to the pathogenesis of Opitz BBB/G syndrome. BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. 2003; 25:356-366
Schweiger S, Foerster J, Lehmann T, Suckow V, Muller YA, Walter G, et al. The Opitz syndrome gene product, MID1, associates with microtubules. Proceedings of the National Academy of Sciences of the United States of America. 1999; 96:2794-2799
Lindskog S. Structure and mechanism of carbonic anhydrase. Pharmacology & Therapeutics. 1997; 74:1-20
Bertini I, Luchinat C. The structure of cobalt(II)-substituted carbonic anhydrase and its implications for the catalytic mechanism of the enzyme. Annals of the New York Academy of Sciences. 1984; 429:89-98
Tripp BC, Smith K, Ferry JG. Carbonic anhydrase: New insights for an ancient enzyme. The Journal of Biological Chemistry. 2001; 276:48615-48618
Kostic M, Matt T, Martinez-Yamout MA, Dyson HJ, Wright PE. Solution structure of the Hdm2 C2H2C4 RING, a domain critical for ubiquitination of p53. Journal of Molecular Biology. 2006; 363:433-450
Plechanovova A, Jaffray EG, McMahon SA, Johnson KA, Navratilova I, Naismith JH, et al. Mechanism of ubiquitylation by dimeric RING ligase RNF4. Nature Structural & Molecular Biology. 2011; 18:1052-1059
Liew CW, Sun H, Hunter T, Day CL. RING domain dimerization is essential for RNF4 function. The Biochemical Journal. 2010; 431:23-29
Mace PD, Linke K, Feltham R, Schumacher FR, Smith CA, Vaux DL, et al. Structures of the cIAP2 RING domain reveal conformational changes associated with ubiquitin-conjugating enzyme (E2) recruitment. The Journal of Biological Chemistry. 2008; 283:31633-31640
Wagner JM, Roganowicz MD, Skorupka K, Alam SL, Christensen D, Doss G, et al. Mechanism of B-box 2 domain-mediated higher-order assembly of the retroviral restriction factor TRIM5alpha. eLife. 2016; 5:1-26. e16309
Diaz-Griffero F, Li X, Javanbakht H, Song B, Welikala S, Stremlau M, et al. Rapid turnover and polyubiquitylation of the retroviral restriction factor TRIM5. Virology. 2006; 349:300-315
Li X, Sodroski J. The TRIM5alpha B-box 2 domain promotes cooperative binding to the retroviral capsid by mediating higher-order self-association. Journal of Virology. 2008; 82:11495-11502
Cao T, Borden KL, Freemont PS, Etkin LD. Involvement of the rfp tripartite motif in protein-protein interactions and subcellular distribution. Journal of Cell Science. 1997; 110(Pt 14):1563-1571
Pickart CM. Mechanisms underlying ubiquitination. Annual Review of Biochemistry. 2001; 70:503-533
Pickart CM. Ubiquitin biology: An old dog learns an old trick. Nature Cell Biology. 2000; 2:E139-E141
Callis J. The ubiquitination machinery of the ubiquitin system. Arabidopsis Book. 2014; 12:e0174
Darwin KH. Prokaryotic ubiquitin-like protein (pup), proteasomes and pathogenesis. Nature Reviews Microbiology. 2009; 7:485-491
Pickart CM. Ubiquitin in chains. Trends in Biochemical Sciences. 2000; 25:544-548
Akutsu M, Dikic I, Bremm A. Ubiquitin chain diversity at a glance. Journal of Cell Science. 2016; 129:875-880
Nishikawa H, Ooka S, Sato K, Arima K, Okamoto J, Klevit RE, et al. Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase. The Journal of Biological Chemistry. 2004; 279:3916-3924
Kravtsova-Ivantsiv Y, Sommer T, Ciechanover A. The lysine48-based polyubiquitin chain proteasomal signal: Not a single child anymore. Angewandte Chemie. 2013; 52:192-198
Yau R, Rape M. The increasing complexity of the ubiquitin code. Nature Cell Biology. 2016; 18:579-586
Herhaus L, Dikic I. Expanding the ubiquitin code through post-translational modification. EMBO Reports. 2015; 16:1071-1083
Komander D, Clague MJ, Urbe S. Breaking the chains: Structure and function of the deubiquitinases. Nature Reviews Molecular Cell Biology. 2009; 10:550-563
Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: Destruction for the sake of construction. Physiological Reviews. 2002; 82:373-428
Deng L, Wang C, Spencer E, Yang L, Braun A, You J, et al. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell. 2000; 103:351-361
Hofmann RM, Pickart CM. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell. 1999; 96:645-653
Kaiser P, Flick K, Wittenberg C, Reed SI. Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCF(Met30)-mediated inactivation of the transcription factor Met4. Cell. 2000; 102:303-314
Hicke L. Protein regulation by monoubiquitin. Nature Reviews Molecular Cell Biology. 2001; 2:195-201
Metzger MB, Pruneda JN, Klevit RE, Weissman AM. RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination. Biochimica et Biophysica Acta. 2014; 1843:47-60
Kim J, Tipper C, Sodroski J. Role of TRIM5alpha RING domain E3 ubiquitin ligase activity in capsid disassembly, reverse transcription blockade, and restriction of simian immunodeficiency virus. Journal of Virology. 2011; 85:8116-8132
Spratt DE, Wu K, Kovacev J, Pan ZQ , Shaw GS. Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase. The Journal of Biological Chemistry. 2012; 287:17374-17385
Kwon YT, Ciechanover A. The ubiquitin code in the ubiquitin-proteasome system and autophagy. Trends in Biochemical Sciences. 2017; 42:873-886
Christensen DE, Brzovic PS, Klevit RE. E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages. Nature Structural & Molecular Biology. 2007; 14:941-948
Plechanovova A, Jaffray EG, Tatham MH, Naismith JH, Hay RT. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature. 2012; 489:115-120
Soss SE, Klevit RE, Chazin WJ. Activation of UbcH5c~Ub is the result of a shift in interdomain motions of the conjugate bound to U-box E3 ligase E4B. Biochemistry. 2013; 52:2991-2999
Pruneda JN, Littlefield PJ, Soss SE, Nordquist KA, Chazin WJ, Brzovic PS, et al. Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases. Molecular Cell. 2012; 47:933-942
Ulrich HD. Protein-protein interactions within an E2-RING finger complex. Implications for ubiquitin-dependent DNA damage repair. The Journal of Biological Chemistry. 2003; 278:7051-7058
Han X, Du H, Massiah MA. Detection and characterization of the in vitro e3 ligase activity of the human MID1 protein. Journal of Molecular Biology. 2011; 407:505-520
Xia Y, Pao GM, Chen HW, Verma IM, Hunter T. Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein. The Journal of Biological Chemistry. 2003; 278:5255-5263
Wu-Baer F, Lagrazon K, Yuan W, Baer R. The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin. The Journal of Biological Chemistry. 2003; 278:34743-34746
Brzovic PS, Rajagopal P, Hoyt DW, King MC, Klevit RE. Structure of a BRCA1-BARD1 heterodimeric RING-RING complex. Nature Structural Biology. 2001; 8:833-837
Mallery DL, Vandenberg CJ, Hiom K. Activation of the E3 ligase function of the BRCA1/BARD1 complex by polyubiquitin chains. The EMBO Journal. 2002; 21:6755-6762
Linares LK, Hengstermann A, Ciechanover A, Muller S, Scheffner M. HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53. Proceedings of the National Academy of Sciences of the United States of America. 2003; 100:12009-12014
Feltham R, Bettjeman B, Budhidarmo R, Mace PD, Shirley S, Condon SM, et al. Smac mimetics activate the E3 ligase activity of cIAP1 protein by promoting RING domain dimerization. The Journal of Biological Chemistry. 2011; 286:17015-17028
Buchwald G, van der Stoop P, Weichenrieder O, Perrakis A, van Lohuizen M, Sixma TK. Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b. The EMBO Journal. 2006; 25:2465-2474
Bourgeois-Daigneault MC, Thibodeau J. Autoregulation of MARCH1 expression by dimerization and autoubiquitination. Journal of Immunology. 2012; 188:4959-4970
Fu TM, Shen C, Li Q , Zhang P, Wu H. Mechanism of ubiquitin transfer promoted by TRAF6. Proceedings of the National Academy of Sciences of the United States of America. 2018; 115:1783-1788
Bell JL, Malyukova A, Holien JK, Koach J, Parker MW, Kavallaris M, et al. TRIM16 acts as an E3 ubiquitin ligase and can heterodimerize with other TRIM family members. PLoS One. 2012; 7:e37470
Zheng Q , Hou J, Zhou Y, Yang Y, Xie B, Cao X. Siglec1 suppresses antiviral innate immune response by inducing TBK1 degradation via the ubiquitin ligase TRIM27. Cell Research. 2015; 25:1121-1136
Du H, Huang Y, Zaghlula M, Walters E, Cox TC, Massiah MA. The MID1 E3 ligase catalyzes the polyubiquitination of Alpha4 (alpha4), a regulatory subunit of protein phosphatase 2A (PP2A): Novel insights into MID1-mediated regulation of PP2A. The Journal of Biological Chemistry. 2013; 288:21341-21350
Koegl M, Hoppe T, Schlenker S, Ulrich HD, Mayer TU, Jentsch S. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell. 1999; 96:635-644
Baranes-Bachar K, Levy-Barda A, Oehler J, Reid DA, Soria-Bretones I, Voss TC, et al. The ubiquitin E3/E4 ligase UBE4A adjusts protein Ubiquitylation and accumulation at sites of DNA damage, facilitating double-Strand break repair. Molecular Cell. 2018; 69:866-878 e7
Ferreira RT, Menezes RA, Rodrigues-Pousada C. E4-ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif. Biology Open. 2015; 4:1122-1131
Chatterjee A, Upadhyay S, Chang X, Nagpal JK, Trink B, Sidransky D. U-box-type ubiquitin E4 ligase, UFD2a attenuates cisplatin mediated degradation of DeltaNp63alpha. Cell Cycle. 2008; 7:1231-1237
Aravind L, Koonin EV. The U box is a modified RING finger—A common domain in ubiquitination. Current Biology. 2000; 10:R132-R134
Ohi MD, Vander Kooi CW, Rosenberg JA, Chazin WJ, Gould KL. Structural insights into the U-box, a domain associated with multi-ubiquitination. Nature Structural Biology. 2003; 10:250-255
Nordquist KA, Dimitrova YN, Brzovic PS, Ridenour WB, Munro KA, Soss SE, et al. Structural and functional characterization of the monomeric U-box domain from E4B. Biochemistry. 2010; 49:347-355
Klevit RE, Herriott JR, Horvath SJ. Solution structure of a zinc finger domain of yeast ADR1. Proteins. 1990; 7:215-226
Kudryashova E, Kudryashov D, Kramerova I, Spencer MJ. Trim32 is a ubiquitin ligase mutated in limb girdle muscular dystrophy type 2H that binds to skeletal muscle myosin and ubiquitinates actin. Journal of Molecular Biology. 2005; 354:413-424
Koliopoulos MG, Esposito D, Christodoulou E, Taylor IA, Rittinger K. Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity. The EMBO Journal. 2016; 35:1204-1218
Javanbakht H, Diaz-Griffero F, Stremlau M, Si Z, Sodroski J. The contribution of RING and B-box 2 domains to retroviral restriction mediated by monkey TRIM5alpha. The Journal of Biological Chemistry. 2005; 280:26933-26940
Gack MU, Shin YC, Joo CH, Urano T, Liang C, Sun L, et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature. 2007; 446:916-920
Villarroya-Beltri C, Guerra S, Sanchez-Madrid F. ISGylation - a key to lock the cell gates for preventing the spread of threats. Journal of Cell Science. 2017; 130:2961-2969
Trockenbacher A, Suckow V, Foerster J, Winter J, Krauss S, Ropers HH, et al. MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation. Nature Genetics. 2001; 29:287-294
Schweiger S, Dorn S, Fuchs M, Kohler A, Matthes F, Muller EC, et al. The E3 ubiquitin ligase MID1 catalyzes ubiquitination and cleavage of Fu. The Journal of Biological Chemistry. 2014; 289:31805-31817
Seshacharyulu P, Pandey P, Datta K, Batra SK. Phosphatase: PP2A structural importance, regulation and its aberrant expression in cancer. Cancer Letters. 2013; 335:9-18
Garcia A, Cayla X, Guergnon J, Dessauge F, Hospital V, Rebollo MP, et al. Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis. Biochimie. 2003; 85:721-726
Janssens V, Goris J. Protein phosphatase 2A: A highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. The Biochemical Journal. 2001; 353:417-439
Sontag E. Protein phosphatase 2A: The Trojan horse of cellular signaling. Cellular Signalling. 2001; 13:7-11
Lechward K, Awotunde OS, Swiatek W, Muszynska G. Protein phosphatase 2A: Variety of forms and diversity of functions. Acta Biochimica Polonica. 2001; 48:921-933
Hunter T. Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signaling. Cell. 1995; 80:225-236
Millward TA, Zolnierowicz S, Hemmings BA. Regulation of protein kinase cascades by protein phosphatase 2A. Trends in Biochemical Sciences. 1999; 24:186-191
Shenolikar S. Protein serine/threonine phosphatases--new avenues for cell regulation. Annual Review of Cell Biology. 1994; 10:55-86
Wera S, Hemmings BA. Serine/threonine protein phosphatases. The Biochemical Journal. 1995; 311(Pt 1):17-29
Cowley S, Paterson H, Kemp P, Marshall CJ. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell. 1994; 77:841-852
Chuang E, Fisher TS, Morgan RW, Robbins MD, Duerr JM, Vander Heiden MG, et al. The CD28 and CTLA-4 receptors associate with the serine/threonine phosphatase PP2A. Immunity. 2000; 13:313-322
Inui S, Kuwahara K, Mizutani J, Maeda K, Kawai T, Nakayasu H, et al. Molecular cloning of a cDNA clone encoding a phosphoprotein component related to the Ig receptor-mediated signal transduction. Journal of Immunology. 1995; 154:2714-2723
Murata K, Wu J, Brautigan DL. B cell receptor-associated protein alpha-4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. Proceedings of the National Academy of Sciences of the United States of America. 1997; 94:10624-10629
Inui S, Sanjo H, Maeda K, Yamamoto H, Miyamoto E, Sakaguchi N. Ig receptor binding protein 1 (alpha4) is associated with a rapamycin-sensitive signal transduction in lymphocytes through direct binding to the catalytic subunit of protein phosphatase 2A. Blood. 1998; 92:539-546
Onda M, Inui S, Maeda K, Suzuki M, Takahashi E, Sakaguchi N. Expression and chromosomal localization of the human alpha 4/IGBP1 gene, the structure of which is closely related to the yeast TAP42 protein of the rapamycin-sensitive signal transduction pathway. Genomics. 1997; 46:373-378
Cygnar KD, Gao X, Pan D, Neufeld TP. The phosphatase subunit tap42 functions independently of target of rapamycin to regulate cell division and survival in Drosophila. Genetics. 2005; 170:733-740
Jiang Y, Broach JR. Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. The EMBO Journal. 1999; 18:2782-2792
Dutcher JP. Mammalian target of rapamycin (mTOR) inhibitors. Current Oncology Reports. 2004; 6:111-115
Mita MM, Mita A, Rowinsky EK. Mammalian target of rapamycin: A new molecular target for breast cancer. Clinical Breast Cancer. 2003; 4:126-137
Rao RD, Buckner JC, Sarkaria JN. Mammalian target of rapamycin (mTOR) inhibitors as anti-cancer agents. Current Cancer Drug Targets. 2004; 4:621-635
Smetana JH, Oliveira CL, Jablonka W, Aguiar Pertinhez T, Carneiro FR, Montero-Lomeli M, et al. Low resolution structure of the human alpha4 protein (IgBP1) and studies on the stability of alpha4 and of its yeast ortholog Tap42. Biochimica et Biophysica Acta. 2006; 1764:724-734
McConnell JL, Watkins GR, Soss SE, Franz HS, McCorvey LR, Spiller BW, et al. Alpha4 is a ubiquitin-binding protein that regulates protein serine/threonine phosphatase 2A ubiquitination. Biochemistry. 2010; 49:1713-1718
Watkins GR, Wang N, Mazalouskas MD, Gomez RJ, Guthrie CR, Kraemer BC, et al. Monoubiquitination promotes calpain cleavage of the protein phosphatase 2A (PP2A) regulatory subunit alpha4, altering PP2A stability and microtubule-associated protein phosphorylation. The Journal of Biological Chemistry. 2012; 287:24207-24215
Fukumoto T, Watanabe-Fukunaga R, Fujisawa K, Nagata S, Fukunaga R. The fused protein kinase regulates hedgehog-stimulated transcriptional activation in Drosophila Schneider 2 cells. The Journal of Biological Chemistry. 2001; 276:38441-38448
Peng H, Begg GE, Schultz DC, Friedman JR, Jensen DE, Speicher DW, et al. Reconstitution of the KRAB-KAP-1 repressor complex: A model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions. Journal of Molecular Biology. 2000; 295:1139-1162
Koyama S, Hata S, Witt CC, Ono Y, Lerche S, Ojima K, et al. Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis. Journal of Molecular Biology. 2008; 376:1224-1236
Mrosek M, Meier S, Ucurum-Fotiadis Z, von Castelmur E, Hedbom E, Lustig A, et al. Structural analysis of B-box 2 from MuRF1: Identification of a novel self-association pattern in a RING-like fold. Biochemistry. 2008; 47:10722-10730
Wright KM, Du H, Massiah MA. Structural and functional observations of the P151L MID1 mutation reveal alpha4 plays a significant role in X-linked Opitz syndrome. The FEBS Journal. 2017; 284:2183-2193
Hodson C, Purkiss A, Miles JA, Walden H. Structure of the human FANCL RING-Ube2T complex reveals determinants of cognate E3-E2 selection. Structure. 2014; 22:337-344
Huang SY, Naik MT, Chang CF, Fang PJ, Wang YH, Shih HM, et al. The B-box 1 dimer of human promyelocytic leukemia protein. Journal of Biomolecular NMR. 2014; 60:275-281
Du H, Massiah MA. NMR studies of the C-terminus of alpha4 reveal possible mechanism of its interaction with MID1 and protein phosphatase 2A. PLoS One. 2011; 6:e28877
Dickson C, Fletcher AJ, Vaysburd M, Yang JC, Mallery DL, Zeng J, et al. Intracellular antibody signalling is regulated by phosphorylation of the fc receptor TRIM21. eLife. 2018; 7:1-22. e32660
Wallenhammar A, Anandapadamanaban M, Lemak A, Mirabello C, Lundstrom P, Wallner B, et al. Solution NMR structure of the TRIM21 B-box2 and identification of residues involved in its interaction with the RING domain. PLoS One. 2017; 12:e0181551
Hatakeyama S. TRIM family proteins: Roles in autophagy, immunity, and carcinogenesis. Trends in Biochemical Sciences. 2017; 42:297-311
Gumucio DL, Diaz A, Schaner P, Richards N, Babcock C, Schaller M, et al. Fire and ICE: The role of pyrin domain-containing proteins in inflammation and apoptosis. Clinical and Experimental Rheumatology. 2002; 20:S45-S53
Yu JW, Fernandes-Alnemri T, Datta P, Wu J, Juliana C, Solorzano L, et al. Pyrin activates the ASC pyroptosome in response to engagement by autoinflammatory PSTPIP1 mutants. Molecular Cell. 2007; 28:214-227
Hatakeyama S. Early evidence for the role of TRIM29 in multiple cancer models. Expert Opinion on Therapeutic Targets. 2016; 20:767-770
Henderson BR. Regulation of BRCA1, BRCA2 and BARD1 intracellular trafficking. BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. 2005; 27:884-893
Sho T, Tsukiyama T, Sato T, Kondo T, Cheng J, Saku T, et al. TRIM29 negatively regulates p53 via inhibition of Tip60. Biochimica et Biophysica Acta. 2011; 1813:1245-1253
Xing J, Weng L, Yuan B, Wang Z, Jia L, Jin R, et al. Identification of a role for TRIM29 in the control of innate immunity in the respiratory tract. Nature Immunology. 2016; 17:1373-1380
Allen MD, Bycroft M. The solution structure of the ZnF UBP domain of USP33/VDU1. Protein Science: A Publication of the Protein Society. 2007; 16:2072-2075
Chen Z, Lin TC, Bi X, Lu G, Dawson BC, Miranda R, et al. TRIM44 promotes quiescent multiple myeloma cell occupancy and survival in the osteoblastic niche via HIF-1alpha stabilization. Leukemia. 2018
Su X, Wang J, Chen W, Li Z, Fu X, Yang A. Overexpression of TRIM14 promotes tongue squamous cell carcinoma aggressiveness by activating the NF-kappaB signaling pathway. Oncotarget. 2016; 7:9939-9950
Zhou Z, Jia X, Xue Q , Dou Z, Ma Y, Zhao Z, et al. TRIM14 is a mitochondrial adaptor that facilitates retinoic acid-inducible gene-I-like receptor-mediated innate immune response. Proceedings of the National Academy of Sciences of the United States of America. 2014; 111:E245-E254
Dai HY, Ma Y, Da Z, Hou XM. Knockdown of TRIM66 inhibits malignant behavior and epithelial-mesenchymal transition in non-small cell lung cancer. Pathology, Research and Practice. 2018; 214:1130-1135
Khanna R, Kronmiller B, Maszle DR, Coupland G, Holm M, Mizuno T, et al. The Arabidopsis B-box zinc finger family. The Plant Cell. 2009; 21:3416-3420
Zou ZY, Wang RH, Wang R, Yang SL, Yang YJ. Genome-wide identification, phylogenetic analysis, and expression profiling of the BBX family genes in pear. The Journal of Horticultural Science and Biotechnology. 2018; 93:37-50
Huang J, Zhao X, Weng X, Wang L, Xie W. The rice B-box zinc finger gene family: Genomic identification, characterization, expression profiling and diurnal analysis. PLoS One. 2012; 7:e48242
Gangappa SN, Botto JF. The BBX family of plant transcription factors. Trends in Plant Science. 2014; 19:460-470
Xu D, Jiang Y, Li J, Holm M, Deng XW. The B-box domain protein BBX21 promotes Photomorphogenesis. Plant Physiology. 2018; 176:2365-2375
Xu D, Jiang Y, Li J, Lin F, Holm M, Deng XW. BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation. Proceedings of the National Academy of Sciences of the United States of America. 2016; 113:7655-7660
Gangappa SN, Crocco CD, Johansson H, Datta S, Hettiarachchi C, Holm M, et al. The Arabidopsis B-BOX protein BBX25 interacts with HY5, negatively regulating BBX22 expression to suppress seedling photomorphogenesis. The Plant Cell. 2013; 25:1243-1257
Gangappa SN, Holm M, Botto JF. Molecular interactions of BBX24 and BBX25 with HYH, HY5 HOMOLOG, to modulate Arabidopsis seedling development. Plant Signaling & Behavior. 2013; 8(8):1-4. e25208