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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biosci Trends. Author manuscript; available in PMC 2010 May 4.
Published in final edited form as:
Biosci Trends. 2007 August; 1(1): 16–25.
PMCID: PMC2862990

The enzymes in ubiquitin-like post-translational modifications


Ubiquitin and at least ten ubiquitin-like proteins are important post-translational modifiers that regulate nearly every aspect of cellular function. These modifications require several chemical reactions that are catalyzed by at least three enzymes. Significant progress has been made in the structure-function analysis of these enzymes. This review describes new advancements in an understanding of the mechanisms of the enzymes catalyzing ubiquitin-like modifications, and highlights the important problems that remain to be addressed.

Keywords: SUMO, ubiquitin, enzymes, X-ray crystallography, NMR spectroscopy


The discovery that ubiquitin can conjugate to target proteins to regulate their cellular life spans and functions has revolutionized our understanding of eukaryotic regulation (1,2). Ubiquitin belongs to a family of at least ten homologous protein modifiers that conjugate to cellular target proteins using a similar biochemical mechanism (3,4). Post-translational modifications with these ubiquitin-like modifiers regulate nearly every aspect of cellular functions including immune response, viral and bacterial infection, gene transcription, RNA processing, DNA-repair, cell cycle progression, and intracellular trafficking.

Ubiquitin-like modifications are different from other post-translational modifications in that they require multiple enzymes to catalyze several sequential reactions (5). The chemical reactions leading to ubiquitination are adenylation, thioester formation, transesterification and isopeptide bond formation. Catalysis is strictly necessary for the adenylation step; the other reactions can occur in the absence of enzymes, but at much slower rates. For example, an intein-based method for protein ligation in vitro, which proceeds via similar chemistry as ubiquitination, can take as long as overnight to complete at room temperature (6). Other intracellular processes, such as protein lipidation and non-ribosomal peptide synthesis also use similar, catalyzed chemical mechanisms (7,8). The enzymes in all of these processes dramatically accelerate the reactions by mechanisms that are not yet well understood.

The ubiquitin-like modifications universally require at least four types of enzymes referred to generally as isopeptidase, E1 (activation enzyme), E2 (conjugation enzyme) and E3 (ligase) (Figure 1). A ubiquitin-like protein (Ublp) is usually synthesized as a precursor, which is matured by an isopeptidase to remove C-terminal residues and expose the Gly-Gly motif. Conjugation of a Ublp to target proteins then begins with E1, which catalyzes the adenylation of Ublp’s C-terminal COOH group. The adenylated Ublp binds E1 non-covalently, and a thioester bond is formed between the SH group of a Cys residue on E1 and the C-terminal -COOH group of Ublp. Ublp is then transferred to a conjugation enzyme E2, where it forms a thioester bond with the -SH group of the catalytic Cys residue of the E2. In the final step, Ublp is attached to target proteins by the formation of an isopeptide bond between its C-terminal -COOH group and the ε-amino group of a Lys residue on the target protein. This step generally requires E3 ligase, although additional protein factors referred to as E4 may also be involved in poly-ubiquitination of some proteins (9). The E1, E2 and E3 enzymes are commonly involved in nearly all ubiquitin-like modifications. Isopeptidases also remove ubiquitin-like proteins from modified targets, and thus regulate the levels of these modifications.

Figure 1
Schematic diagram of the chemical reactions involved in ubiquitin-like modifications.

The ubiquitin-like post-translational modifications are dependent on protein-protein interactions between the enzymes, protein substrates, and Ublp to accomplish each step of the reactions leading to the modifications. The protein associations in these processes are dynamic, and a stable complex of all components does not exist (10). Characterizing the molecular mechanism of these multi-enzymatic processes is important to our understanding of how multi-protein machineries carry out macromolecular chemistry. This review will focus on the recent structural and enzymological studies of the enzymes in ubiquitin-like post-translational modifications.

The E1 enzyme and the transfer of Ublp from E1 to E2

A single and unique E1 enzyme is responsible for activating each ubiquitin-like modification (11). Both the ubiquitin and SUMO E1 enzymes are essential genes in yeast (12,13). The three-dimensional structures of the NEDD8 and SUMO E1 enzymes as well as several of their complexes have been solved (14,15). The structure of a small domain of ubiquitin E1 has also been solved (16). These E1 enzymes contain regions that resemble the bacterial proteins ThioS and MoeB (1720). Both the SUMO and NEDD8 E1 enzymes are tight heterodimers of two polypeptides, which are homologous to the N-terminal and C-terminal portions of the ubiquitin E1, respectively. The overall structure of E1 contains three domains (Figure 2A). One domain contains the ATP-binding site and catalyzes the adenylation of Ublp. Another domain contains the catalytic Cys residue which forms a covalent thioester bond with the C-terminus of a Ublp. The third domain has a three-dimensional fold similar to that of ubiquitin in the absence of any sequence similarity, and is known as the Ubl domain.

Figure 2
Summary of structural mechanism of E1. (A) The structure of NEDD8 E1 with different domains color as red, blue and green corresponding to the Ubl, Cys, and adenylation domains, respectively. The catalytic Cys is indicated in orange on the Cys domain. ...

Recently structural studies using both X-ray crystallography and NMR spectroscopy have greatly advanced our understanding of the mechanism of Ublp’s transfer from E1 to E2. The Ubl domain, Cys domain and adenylation domain of E1 all participate in recruiting E2 for the transfer of Ublp from E1 to E2 (21,22) (Figure 2C). The Ubl domain has the highest affinity for E2 among the three E1 domains (23). It also has the flexibility to undergo a large scale rotation to properly position E2 (Figures 2B and 2C) (22). In addition, our NMR study has shown that the SUMO E2 has an intrinsic affinity for the Cys domain of its cognate E1 (Figure 2D) (21). The affinity between the E2 and the Cys domain of E1 is not high, but is important for the guided translocation of E2 to the catalytic Cys residue of E1 and for properly positioning the E2 for the transfer of SUMO from E1 to E2. The affinity between E2 and the adenylation domain is also expected to be weak, based on the small contact interface. The multiple low affinity binding sites on E1 for E2 provide an effective high affinity between the two enzymes to ensure efficient catalysis at low protein concentrations. At the same time, the low affinity of each site allows rapid protein association and dissociation for efficient catalysis.

The mechanism of how an Ublp translocates from the adenylation active site to catalytic Cys on E1 remains unclear. The structure of NEDD8 E1 in complex with NEDD8 and ATP, and the structure of SUMO E1 in complex with SUMO-1 and ATP have shown similar features of how the adenylated Ublps bind to their cognate E1 (14,15). The C-termini of both NEDD8 and SUMO are buried deeply within their cognate E1 enzymes near the adenylation active site. However, this site is distal (approximately 30 Å away) from the Cys residues with which it forms the thioester bonds in the subsequent step of the conjugation pathway (Figure 2E). The structures raise the question of how Ublp transfers with high efficiency and specificity between the two catalytic active sites of E1.

The E2 enzyme and its recognition of substrates and Ublp

Multiple E2 enzymes have been identified for ubiquitination of different target proteins (1), but only one specific E2 appears to be required by each of the ubiquitin-like proteins NEDD8 (24) and ISG15 (25). Additionally, a single E2 called Ubc9 serves all SUMO paralogues (26). The E2 enzymes are of variable sizes, but they all contain a core catalytic domain of approximately 150 amino acid residues (Figure 3). Some E2s consist of just the core catalytic domain, whereas others contain N- and/or C-terminal extensions of variable lengths beyond their core catalytic domains (27). There are also E2-like proteins involved in ubiquitin-dependent processes that lack the catalytic Cys residues and are catalytically inactive, such as the Tsg101 UEV domain and Mms2 (28,29).

Figure 3
Summary of current knowledge of key functional sites of E2. The structure of Ubc9, shown in orange, is a representative of the conserved catalytic core structure of E2. The different functional sites are indicated in the figure. The thioester bound Ublp ...

The core catalytic domains of E2 enzymes have a highly conserved three-dimensional structure, with the biggest differences manifested in two surface loops of variable lengths (3033). One of the variable loop insertions in E2 is located adjacent to the catalytic Cys residue (Figure 3). Mutations in this loop of Ubc9 affected the transfer of SUMO from E1 to E2 (34). Enzyme kinetic analysis indicates that this loop is also important for substrate recognition by the E2. A conserved Asn residue near the active site Cys has been identified as the catalytic residue that stabilizes the transition state oxyanion during the transfer of Ublp from E2 to target proteins (35).

Characterization of the SUMO modification pathway has provided a clear indication of direct substrate-E2 interaction. A consensus sequence, ψKxE (where ψ represents a bulky hydrophobic residue, K is a Lys, x is any amino acid, and E is a Glu), occurs in most SUMO-1 substrate proteins (36), although modifications at non-consensus sequences may occur much less frequently (37). This consensus amino acid sequence in substrates is a Ubc9 binding motif, and binds specifically to a region of Ubc9 near the active site Cys93 (Figure 3) (38,39). The substrate-binding surface has been previously shown to include residues that demonstrate significant dynamics on the microsecond to millisecond time scale (40). The conformational flexibility in E2 appears to be conserved, because the region of Ubc7 that is equivalent to Ubc9’s substrate binding site also has one of the largest structural variations between crystal structures of the same protein determined from different complexes (41,42). The Lys residue where the modification occurs on substrates is found in a groove formed by the backbone atoms of residues Asp127, Pro128, Ala129 and the sidechain atoms of residues Asp127 and Tyr87 of Ubc9. In this position, the ε-amino group of the substrate Lys is 2.6 Å away from the Sγ atom of Ubc9’s Cys93. Among the four residues that form important contacts with the Lys at the modification site, only the Pro and Ala are relatively conserved in E2 enzymes catalyzing ubiquitination. It remains to be established that a similar substrate binding mechanism is involved in the ubiquitination pathway; however, ubiquitination sites of target proteins do not have a consensus sequence. The direct interaction of substrates and Ubc9 may account for the lack of HECT-domain containing E3s in SUMO modifications.

Ubiquitin E2 enzymes have been shown to form homo and heterodimers. For example, Ubc3 (CDC34) forms a heterodimer with the DNA repair related Ubc2 (Rad6) (43) and also forms a homodimer facilitated by the formation of ubiquitin thioester (44). These dimers are required for the degradation of many key regulators of cell cycle progression through the proteosome pathway. For example, a Ubc6 and Ubc7 heterodimer is responsible for the turnover of Matα2 transcription factor in yeast (45). Dimerization of E2s may be responsible for the formation of poly-ubiquitin chains in some cases; however, dimerization does not appear to be a general requirement for poly-ubiquitination. For example, Ubc5 does not appear to form homo- or heterodimers in vitro or in vivo (40), but Ubc5 can catalyze the formation of poly-ubiquitin chains (45).

Ublp-E2 thioester is an important intermediate in ubiquitin-like conjugation. Biochemical studies have shown that different ubiquitin-E2 thioester have different stabilities (46). Several structural characterizations of ubiquitin-E2 thioester covalent complexes have been carried out using NMR methods. In one study, the catalytic Cys residue was mutated to a Ser to produce an Ubc2b-ubiquitin oxyester, which has a more stable covalent linkage than thioester, and the oxyester was purified for NMR characterization (47). In other studies, productive thioesters were made in the NMR tube by the addition of E1 and ATP (48,49). These studies have shown that thioester bound ubiquitin contacts the surface of E2 that is centered on the second α-helix, which is adjacent to the active site Cys (Figure 3). The binding interface on ubiquitin is located on the β-sheet. The first study also demonstrated that the oxyester-bonded ubiquitin has linewidths that are similar to that of the free protein, and this suggests that ubiquitin moves somewhat independently from the covalently bound E2.

A specific and conserved non-covalent interaction between Ublp and E2 has been observed across the ubiquitin-like proteins (23,47,4951). The non-covalent interaction between SUMO-1 and Ubc9 involves interfaces that are structurally conserved in Ublps and E2 enzymes. The binding site on Ubc9 covers the N-terminal helix, following β-strand, and the loop between them (Figure 3), and the binding surface on SUMO-1 is located on the main β-sheet (50). This interaction is conserved across SUMO paralogues, and is both enthalpically and entropically driven (26). Similar interactions have been observed between an E2 and ubiquitin, between the E2-like protein Mms2 and ubiquitin (47,49,51). However, the ubiquitin-like domains of the NEDD8 and SUMO E1 enzymes bind to their E2 in a similar, but clearly distinct manner (23,52). The functions of the non-covalent Ublp-E2 interaction are unclear. Mutations in human Ubc9 that disrupt the interaction with SUMO were shown to significantly reduce the transfer rate of SUMO-1 from E1 to E2 without affecting the transfer of SUMO from E2 to substrates (26). Similar amino acid substitutions on ubiquitin E2 hindered its catalysis of poly-ubiquitin chains (53). Further studies are necessary in order to understand the role of Ublp-E2 interaction.

The E3 and E4 enzymes

E3 ligases have received the most attention among the three enzymes involved in ubiquitin-like modifications, because many E3s are key players in essential cellular processes, and/or are prominent oncogenes and tumor suppressor genes. For example, one of the proteins frequently mutated in breast and ovarian cancers, BRCA1, has E3 ligase activities for both ubiquitination and sumoylation (54). The SCF (Skp1-Cullin-F-box protein) complexes and APC (anaphase-promoting complex), which are critical for cell-cycle progression, are ubiquitin E3 ligases (5558). The host ubiquitin-like modification systems can be hijacked for pathogen infections. For example, the human papilloma virus (HPV) encoded E6 protein and E6-AP (E6 associated protein) form a complex that functions as an E3 ligase to reduce the level of the tumor suppressor protein p53 in HPV positive tumors (59,60). Knowledge about E3 ligases has enabled the development of research tools. For example, over-expression of specific ubiquitin E3 in cells can achieve specific and sustained knockout of only a subset of a target protein (61). Most E3s that catalyze ubiquitin-like modifications contain either the HECT or the RING domain (62). Both the HECT and RING domains are E2 recognition motifs (41,42,6365).

HECT containing E3s receive ubiquitin from E2 to form thioester bonds with ubiquitin before transferring it to substrate proteins. The three-dimensional structures of several HECT domains in complex with E2s have been determined by X-ray crystallography (42,66,67). The HECT domain contains three subdomains; the N-terminal subdomain contacts E2 directly, the C-terminal subdomain contains the catalytic Cys residue, and the middle subdomain separates the two (Figure 4A). The linkers connecting the different subdomains have considerable flexibility, which allows the subdomains to move quite independently. Such domain movement can change the distance between the catalytic Cys residues on HECT and on E2 from 40 Å apart to close enough that the transesterification reaction can happen efficiently (Figure 4A). There is likely a general base to stabilize the transition state in which the HECT thioester bond is broken and the substrate isopeptide bond is formed. However, such a residue has not been identified in the HECT motif.

Figure 4
E3 structures and regulation. (A) The interaction between E2 and HECT motif. E2 is shown in orange and one HECT domain is shown in green. Another HECT domain is also shown in magenta in order to illustrate the conformational flexibility of HECT domains. ...

The RING-containing E3s constitute the largest family of E3 and can be divided into two sub-families, Zn-binding RING and Zn-independent RING-like fold formed by the U-box motif (65,68). RING motifs bind to the same site of E2 as the HECT domain despite the absence of sequence and structural similarity to the HECT domain (Figure 4B). Some U-box containing proteins have been shown to function as E4s, which recognize mono-ubiquitinated proteins and catalyze the formation of long poly-ubiquitin chains (9). RING-containing E3s do not form thioester intermediates with ubiquitin, but bind to both E2 and substrate proteins. Both E3 recognition of substrate proteins and E2 enzymes are critical for catalyzing substrate modifications. For example, E3s in the N-end rule pathways recognize specific N-terminal residues of their target proteins (2). Most SUMO E3 enzymes contain RING domains and belong to the Siz/PIAS family of proteins (6972). These SUMO E3s are similar to RING containing ubiquitin E3s in that they use the RING domains for binding E2 and also contain separate domains for binding target proteins.

Biochemical and structural studies have not provided a clear understanding of how RING-containing E3 ligases activate the transfer of Ublp from E2 to substrates. Because substrate and E2 binding activities are both required for the function of most of these ligases, the RING-containing E3s are thought of as adaptors that bring substrate and E2 together. Structural studies of a SCF complex indicate that the substrate and E2 binding sites on this E3 are surprisingly distal (as far apart as 50 Å) (41,42,63,7376). Such a wide distance between the substrate and E2 was contrary to the efficiency of the E3 (77). In one study, a flexible linker that was engineered in the Cullin protein of a SCF complex destroyed the E3’s enzymatic activity, indicating that the rigidity of SCF complexes is important for their ligase activity (63). This is clearly different from the catalytic mechanism employed by HECT domains. Additionally, there are increasing biochemical data contradicting the theory that these E3s are merely adaptors. For example, it has been shown that a small subunit in APC containing only the RING domain, but not the substrate binding domain, could enhance APC specific ubiquitination (78,79). Another study also showed that simply bringing an E2 and a substrate close together by fusing a substrate protein to the C-terminus of an E2 was not sufficient to bypass E3 to activate ubiquitination of the substrate protein (80). It has also been shown that E3 interacts with E2 differently from interacting with E2-ubiquitin thioester (81,82). However, the structural details of the interaction between E2-ubiquitin thioester and an E3 have not been characterized.

Another mystery in the E3 mechanism is the way in which poly-ubiquitin chains are formed. Poly-ubiquitin chains can form on E2 first and are then transferred to substrates, or ubiquitin is added one by one from an E2 to substrate proteins (83). Thus, the E3 catalyzed reactions have substrate “flexibility”, which is either the target protein or the ubiquitin moiety of the ubiquitinated target protein. On the other hand, the reaction has clear “specificity”; in most cases, the poly-ubiquitin chains are formed on a very specific Lys residue (e.g. Lys48 or Lys63) on ubiquitin, instead of randomly on any Lys residue. The substrate flexibility and specificity in ubiquitination is distinct from most well characterized enzymatic reactions. In some cases, an E3 ligase only catalyzes mono-ubiquitination, and then an E4 takes over to add poly-ubiquitin chains to target proteins (9). However, in most in vitro reactions, poly-ubiquitination readily occurs with the addition of E3 and in the absence of E4.

Compared to the E1 and E2 enzymes, the E3 enzymes are much less conserved. For example, a SUMO E3 RanBP2 (also known as Nup358) (84) do not contain HECT, RING or U-box domains (84). Unlike other characterized E3s, RanBP2 does not form detectable non-covalent complexes with target proteins, nor does it form thioester bonds with SUMO-1 (84). The three-dimensional structure of the region of RanBP2 that has the E3 ligase function in complex with Ubc9 and sumoylated RanGAP1 has been solved by X-ray crystallography (85), which shows that the interaction between Ubc9 and RanBP2 is distinct from previously characterized E2-E3 interactions (Figure 4C). In this case, the E2-E3 interaction involves a surface of E2 that is different from that involved in binding the HECT or RING containing E3s (86). In general, it is not clear how the structurally distinct RING-containing E3s and RanBP2 achieve similar functions in the absence of sequence and structural conservation.

An interesting finding from the RanBP2-related studies is that the interaction between SUMO and E3 is critical for the E3’s ligase activity (85). The M-IR2 domain of RanBP2, which binds specifically to SUMO-1 and not to SUMO-2, can catalyze SUMO-1 but not SUMO-2 modifications (86). This is the first E3 ligase in which the interaction between an E3 ligase and the ubiquitin-like protein has been shown to be important for the ligase activity, although it has been known for more than 20 years that some ubiquitin E3 ligases bind ubiquitin non-covalently (10).

Enzyme kinetic analysis of the conjugation process

Quantitative enzyme kinetic analysis is necessary to provide insights into reaction mechanisms that would be difficult to obtain otherwise. Quantitative enzyme kinetic approaches have been developed to examine every step of the reactions that lead to ubiquitin-like modifications. An elegant quantitative framework has been developed to characterize each step of E1 catalyzed reactions (87). Steady-state methods can be used to obtain enzyme kinetic information for the E2 and E3 enzymes (39,82). These approaches take advantage of the fact that in a reaction requiring multiple enzymes, kinetic information on a particular enzyme can be extracted when this enzyme limits the overall reaction rate. For example, in sumoylation reactions, the conditions can be set so that E2 limits the rate of the overall reactions. Therefore, the kinetic parameters obtained reflect the properties of E2 (39). A non-steady-state kinetic approach (34) was also developed based on the transfer experiments pioneered in studies of the ubiquitin modification pathway (10). The advantage of the non-steady-state kinetic analysis is that one can extract individual kinetic constants, such as on-rate and off-rate of the enzyme-substrate association, and the catalytic rate constant in order to gain detailed insights into each step of the reaction. Fluorescence labeled protein substrates are extremely useful for evaluating the reaction rates (88), particularly in quantitating the heterogeneous products of ubiquitination reactions (89).

The deconjugation enzyme

The deconjugation enzymes in the ubiquitin and homologous pathways have two functions: to activate the Ublp and to remove Ublp from target proteins (90). As mentioned above, the first step in Ublp activation is the cleavage of the C-terminal residues by deconjugation enzymes to expose the Gly-Gly motif. The deconjugation enzymes are also important in regulating Ublp’s modifications by removing these modifications.

The deconjugation enzymes can be classified into different families: the ubiquitin C-terminal hydrolases (UCH) that remove small peptides from the C-terminus of ubiquitin (91), ubiquitin-specific processing proteases (UBP) that remove mono- and poly-ubiquitin modifications (92), the ubiquitin-like proteases (ULP) that act on SUMO and NEDD8 (9396), the JAMM motif-containing metalloproteases that act on ubiquitin and NEDD8 (97), and members of the ovarian tumor (OTU) superfamily (98). The different families of deconjugation enzymes do not share significant sequence similarities and overall folds. Except for the JAMM motif-containing metalloprotease family, the other four families of deconjugation enzymes have conserved geometry of the catalytic triad formed by a His, an Asn and a Cys (99104). They belong to the cysteine protease superfamily. Therefore, inhibitors of the cysteine proteases can also inhibit these deconjugation enzymes. The JAMM motif-containing proteases have diverse functions, including deubiquitination and degradation by the 26S proteasome, and deubiquitination of proteins at the endosome (105).

The deconjugation enzymes present excellent targets for developing research tools and therapeutics because they are critical for regulating these post-translational modifications, and thus directly interfere with related biological functions. For example, the deubiquitination activity of CYLD, which encodes an UBP and has UBP activity, has been shown to play a role in regulating the activity of NF-κB (106). Mutations in CYLD are linked to familial cylindromatosis, which is an autosomal dominant predisposition to tumors of skin appendages. Most cancer associated mutations of CYLD result in truncations or frameshift alterations of the UBP domain. Herpes virus associated ubiquitin-specific protease (HAUSP or USP7) is responsible for deubiquitination of ubiquitinated p53 and promotion of p53 dependent cell growth arrest and apoptosis (107). Dysfunction in UCH has been associated with neurodegeneration and cancer (108). In the human genome, there are many uncharacterized proteins that resemble the sequences of deconjugation enzymes. Their functions as deconjugation enzymes for ubiquitin or ubiquitin-like modifications remain to be established.

Interplay between different post-translational modifications

Ubiquitin-like modifications are regulated by each other and by other post-translational modifications. For example, the ubiquitin-like protein NEDD8 modifies the cullin family of proteins, which are subunits of the SCF family of ubiquitin E3 ligases (55). Sumoylation and ubiquitination sometimes occur at the same Lys residue and antagonize each other (109). The oncogene MDM2, which contains a RING motif, has the ligase activity for both NEDD8 and ubiquitin modifications of the tumor suppressor protein p53 (110). Phosphorylation and dephosphorylation of target proteins can regulate the interactions of target proteins with E3 ligases (reviewed in (62)). Sumoylation of a transcription factor or histone triggers recruitment of histone deacetylases (HDAC) to remove histone acetylation and thus leads to transcriptional repression (111,112). Ubiquitination of histone H2B regulates its methylation and leads to gene silencing (113).

Concluding remarks

Significant progress has been made in elucidating the molecular mechanism of the enzymes involved in ubiquitin and ubiquitin-like modifications. Ubiquitin-like modifications are similar to other intracellular macromolecular chemical reactions, such as DNA repair, gene transcription, and RNA processing, in that they require multiple proteins to catalyze multiple reactions. The common theme in these processes is that the protein-protein interactions often involve multiple, medium to low affinity binding sites. The multivalent, medium to low affinity interactions allow for the rapid turn over of the enzymes and the efficient catalysis at low enzyme and substrate concentrations. Similarly, low affinity, multivalent protein-protein interactions are also common in Ublp-dependent protein-protein interactions. Ubiquitin-like proteins provide additional docking sites for protein-protein interactions. However, the affinities of ubiquitin-binding motifs for ubiquitin or the SUMO-binding motif for SUMO were not high with the Kd in the 10100 μM range (114). Despite the low affinities, these interactions play critical roles in the presence of other low or medium affinity interactions, and offer a mechanism to turn protein-protein interactions on and off quickly by conjugation and deconjugation of Ublps. Ubiquitin-like modifications are important in nearly every aspect of cellular function. Therefore, understanding the mechanism of the enzymes catalyzing these modifications will lead to the development of strategies to manipulate them for developing research tools and novel therapeutic approaches.


1. Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998;67:425–479. [PubMed]
2. Varshavsky A. The ubiquitin system. Trends Biochem Sci. 1997;22:383–387. [PubMed]
3. Wilkinson CR. Ubiquitin-like proteins: meet the family. Semin Cell Dev Biol. 2004;15:199–200. [PubMed]
4. Welchman RL, Gordon C, Mayer RJ. Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat Rev Mol Cell Biol. 2005;6:599–609. [PubMed]
5. Rose IA. Ubiquitin at fox chase. Proc Natl Acad Sci U S A. 2005;102:11575–11577. [PubMed]
6. Blaschke UK, Silberstein J, Muir TW. Protein engineering by expressed protein ligation. Methods Enzymol. 2000;328:478–496. [PubMed]
7. Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi Y, Ishihara N, Mizushima N, Tanida I, Kominami E, Ohsumi M, Noda T, Ohsumi Y. A ubiquitin-like system mediates protein lipidation. Nature. 2000;408:488–492. [PubMed]
8. Marahiel MA, Stachelhaus T, Mootz HD. Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem Rev. 1997;97:2651–2674. [PubMed]
9. 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. [PubMed]
10. Hershko A, Heller H, Elias S, Ciechanover A. Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J Biol Chem. 1983;258:8206–8214. [PubMed]
11. Huang DT, Walden H, Duda D, Schulman BA. Ubiquitin-like protein activation. Oncogene. 2004;23:1958–1971. [PubMed]
12. McGrath JP, Jentsch S, Varshavsky A. UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme. EMBO J. 1991;10:227–236. [PubMed]
13. Dohmen RJ, Stappen R, McGrath JP, Forrova H, Kolarov J, Goffeau A, Varshavsky A. An essential yeast gene encoding a homolog of ubiquitin-activating enzyme. J Biol Chem. 1995;270:18099–18109. [PubMed]
14. Walden H, Podgorski MS, Huang DT, Miller DW, Howard RJ, Minor DL, Holton JM, Schulman BA. The structure of the APPBP1-UBA3-NEDD8-ATP complex reveals the basis for selective ubiquitin-like protein activation by an E1. Mol Cell. 2003;12:1427–1437. [PubMed]
15. Lois LM, Lima CD. Structures of the SUMO E1 provide mechanistic insights into SUMO activation and E2 recruitment to E1. EMBO J. 2005;24:439–451. [PubMed]
16. Szczepanowski RH, Filipek R, Bochtler M. Crystal structure of a fragment of mouse ubiquitin-activating enzyme. J Biol Chem. 2005;280:22006–22011. [PubMed]
17. Duda DM, Walden H, Sfondouris J, Schulman BA. Structural analysis of Escherichia coli ThiF. J Mol Biol. 2005;349:774–786. [PubMed]
18. Lake MW, Wuebbens MM, Rajagopalan KV, Schindelin H. Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB-MoaD complex. Nature. 2001;414:325–329. [PubMed]
19. Xi J, Ge Y, Kinsland C, McLafferty FW, Begley TP. Biosynthesis of the thiazole moiety of thiamin in Escherichia coli: identification of an acyldisulfide-linked protein--protein conjugate that is functionally analogous to the ubiquitin/E1 complex. Proc Natl Acad Sci U S A. 2001;98:8513–8518. [PubMed]
20. Leimkuhler S, Wuebbens MM, Rajagopalan KV. Characterization of Escherichia coli MoeB and its involvement in the activation of molybdopterin synthase for the biosynthesis of the molybdenum cofactor. J Biol Chem. 2001;276:4695–34701. [PubMed]
21. Wang J, Hu W, Cai S, Lee B, Song J, Chen Y. The Intrinsic Affinity between E2 and the Cys Domain of E1 in Ubiquitin-like Modifications. Mol Cell. 2007 in press. [PMC free article] [PubMed]
22. Huang DT, Hunt HW, Zhuang M, Ohi MD, Holton JM, Schulman BA. Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity. Nature. 2007;445:394–398. [PMC free article] [PubMed]
23. Huang DT, Paydar A, Zhuang M, Waddell MB, Holton JM, Schulman BA. Structural basis for recruitment of Ubc12 by an E2 binding domain in NEDD8’s E1. Mol Cell. 2005;17:341–350. [PubMed]
24. Parry G, Estelle M. Regulation of cullin-based ubiquitin ligases by the Nedd8/RUB ubiquitin-like proteins. Semin Cell Dev Biol. 2004;15:221–229. [PubMed]
25. Ritchie KJ, Zhang DE. ISG15: the immunological kin of ubiquitin. Semin Cell Dev Biol. 2004;15:237–246. [PubMed]
26. Tatham MH, Kim S, Yu B, Jaffray E, Song J, Zheng J, Rodriguez MS, Hay RT, Chen Y. Role of an N-terminal site of Ubc9 in SUMO-1, -2, and -3 binding and conjugation. Biochemistry. 2003;42:9959–9969. [PubMed]
27. Jentsch S. The ubiquitin-conjugation system. Annu Rev Genet. 1992;26:179–207. [PubMed]
28. VanDemark AP, Hofmann RM, Tsui C, Pickart CM, Wolberger C. Molecular insights into polyubiquitin chain assembly: crystal structure of the Mms2/Ubc13 heterodimer. Cell. 2001;105:711–720. [PubMed]
29. Pornillos O, Alam SL, Rich RL, Myszka DG, Davis DR, Sundquist WI. Structure and functional interactions of the Tsg101 UEV domain. EMBO J. 2002;21:2397–2406. [PubMed]
30. Cook WJ, Jeffrey LC, Sullivan ML, Vierstra RD. Three-dimensional structure of a ubiquitin-conjugating enzyme (E2) J Biol Chem. 1992;267:15116–15121. [PubMed]
31. Cook WJ, Jeffrey LC, Xu Y, Chau V. Tertiary structures of class I ubiquitin-conjugating enzymes are highly conserved: crystal structure of yeast Ubc4. Biochemistry. 1993;32:13809–13817. [PubMed]
32. Cook WJ, Martin PD, Edwards BF, Yamazaki RK, Chau V. Crystal structure of a class I ubiquitin conjugating enzyme (Ubc7) from Saccharomyces cerevisiae at 2.9 angstroms resolution. Biochemistry. 1997;36:1621–1627. [PubMed]
33. Giraud MF, Desterro JM, Naismith JH. Structure of ubiquitin-conjugating enzyme 9 displays significant differences with other ubiquitin-conjugating enzymes which may reflect its specificity for sumo rather than ubiquitin. Acta Crystallogr D Biol Crystallogr. 1998;54:891–898. [PubMed]
34. Tatham MH, Chen Y, Hay RT. Role of two residues proximal to the active site of Ubc9 in substrate recognition by the Ubc9. SUMO-1 thiolester complex. Biochemistry. 2003;42:3168–3179. [PubMed]
35. Wu PY, Hanlon M, Eddins M, Tsui C, Rogers RS, Jensen JP, Matunis MJ, Weissman AM, Wolberger CP, Pickart CM. A conserved catalytic residue in the ubiquitin-conjugating enzyme family. EMBO J. 2003;22:5241–5250. [PubMed]
36. Rodriguez MS, Dargemont C, Hay RT. SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J Biol Chem. 2001;276:12654–12659. [PubMed]
37. Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 2002;419:135–141. [PubMed]
38. Bernier-Villamor V, Sampson DA, Matunis MJ, Lima CD. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell. 2002;108:345–356. [PubMed]
39. Lin D, Tatham MH, Yu B, Kim S, Hay RT, Chen Y. Identification of a substrate recognition site on Ubc9. J Biol Chem. 2002;277:21740–21748. [PubMed]
40. Liu Q, Yuan YC, Shen B, Chen DJ, Chen Y. Conformational flexibility of a ubiquitin conjugation enzyme (E2) Biochemistry. 1999;38:1415–1425. [PubMed]
41. Zheng N, Wang P, Jeffrey PD, Pavletich NP. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases. Cell. 2000;102:533–539. [PubMed]
42. Huang L, Kinnucan E, Wang G, Beaudenon S, Howley PM, Huibregtse JM, Pavletich NP. Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science. 1999;286:1321–1326. [PubMed]
43. Silver ET, Gwozd TJ, Ptak C, Goebl M, Ellison MJ. A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s. EMBO J. 1992;11:3091–3098. [PubMed]
44. Varelas X, Ptak C, Ellison MJ. Cdc34 self-association is facilitated by ubiquitin thiolester formation and is required for its catalytic activity. Mol Cell Biol. 2003;23:5388–5400. [PMC free article] [PubMed]
45. Chen P, Johnson P, Sommer T, Jentsch S, Hochstrasser M. Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MAT alpha 2 repressor. Cell. 1993;74:357–369. [PubMed]
46. Haas AL, Bright PM. The resolution and characterization of putative ubiquitin carrier protein isozymes from rabbit reticulocytes. J Biol Chem. 1988;263:13258–13267. [PubMed]
47. Miura T, Klaus W, Gsell B, Miyamoto C, Senn H. Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution. J Mol Biol. 1999;290:213–228. [PubMed]
48. Hamilton KS, Ellison MJ, Shaw GS. Identification of the ubiquitin interfacial residues in a ubiquitin-E2 covalent complex. J Biomol NMR. 2000;18:319–327. [PubMed]
49. McKenna S, Moraes T, Pastushok L, Ptak C, Xiao W, Spyracopoulos L, Ellison MJ. An NMR-based model of the ubiquitin-bound human ubiquitin conjugation complex Mms2.Ubc13. The structural basis for lysine 63 chain catalysis. J Biol Chem. 2003;278:13151–13158. [PubMed]
50. Liu Q, Jin C, Liao X, Shen Z, Chen DJ, Chen Y. The binding interface between an E2 (UBC9) and a ubiquitin homologue (UBL1) J Biol Chem. 1999;274:16979–16987. [PubMed]
51. McKenna S, Spyracopoulos L, Moraes T, Pastushok L, Ptak C, Xiao W, Ellison MJ. Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination. J Biol Chem. 2001;276:40120–40126. [PubMed]
52. Duda DM, van Waardenburg RC, Borg LA, McGarity S, Nourse A, Waddell MB, Bjornsti MA, Schulman BA. Structure of a SUMO-binding-motif Mimic Bound to Smt3p-Ubc9p: Conservation of a Non-covalent Ubiquitin-like Protein-E2 Complex as a Platform for Selective Interactions within a SUMO Pathway. J Mol Biol. 2007;369:619–630. [PMC free article] [PubMed]
53. Brzovic PS, Lissounov A, Christensen DE, Hoyt DW, Klevit RE. A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination. Mol Cell. 2006;21:873–880. [PubMed]
54. Billack B, Monteiro AN. BRCA1 in breast and ovarian cancer predisposition. Cancer Lett. 2005;227:1–7. [PubMed]
55. Petroski MD, Deshaies RJ. Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol. 2005;6:9–20. [PubMed]
56. Nakayama KI, Nakayama K. Regulation of the cell cycle by SCF-type ubiquitin ligases. Semin Cell Dev Biol. 2005;16:323–333. [PubMed]
57. Pagano M. Cell cycle regulation by the ubiquitin pathway. FASEB J. 1997;13:1067–1075. [PubMed]
58. Willems AR, Schwab M, Tyers M. A hitchhiker’s guide to the cullin ubiquitin ligases: SCF and its kin. Biochim Biophys Acta. 2004;1695:133–170. [PubMed]
59. Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990;248:76–79. [PubMed]
60. Storey A, Thomas M, Kalita A, Harwood C, Gardiol D, Mantovani F, Breuer J, Leigh IM, Matlashewski G, Banks L. Role of a p53 polymorphism in the development of human papillomavirus-associated cancer. Nature. 1998;393:229–234. [PubMed]
61. Zhang J, Zheng N, Zhou P. Exploring the functional complexity of cellular proteins by protein knockout. Proc Natl Acad Sci U S A. 2003;100:14127–14132. [PubMed]
62. Pickart CM. Mechanisms underlying ubiquitination. Annu Rev Biochem. 2001;70:503–533. [PubMed]
63. Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD, Wang P, Chu C, Koepp DM, Elledge SJ, Pagano M, Conaway RC, Conaway JW, Harper JW, Pavletich NP. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature. 2002;416:703–709. [PubMed]
64. Katoh S, Hong C, Tsunoda Y, Murata K, Takai R, Minami E, Yamazaki T, Katoh E. High precision NMR structure and function of the RING-H2 finger domain of EL5, a rice protein whose expression is increased upon exposure to pathogen-derived oligosaccharides. J Biol Chem. 2003;278:15341–15348. [PubMed]
65. Ohi MD, Vander Kooi CW, Rosenberg JA, Chazin WJ, Gould KL. Structural insights into the U-box, a domain associated with multi-ubiquitination. Nat Struct Biol. 2003;10:250–255. [PubMed]
66. Verdecia MA, Joazeiro CA, Wells NJ, Ferrer JL, Bowman ME, Hunter T, Noel JP. Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase. Mol Cell. 2003;11:249–259. [PubMed]
67. Ogunjimi AA, Briant DJ, Pece-Barbara N, Le Roy C, Di Guglielmo GM, Kavsak P, Rasmussen RK, Seet BT, Sicheri F, Wrana JL. Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain. Mol Cell. 2005;19:297–308. [PubMed]
68. Hatakeyama S, Yada M, Matsumoto M, Ishida N, Nakayama KI. U box proteins as a new family of ubiquitin-protein ligases. J Biol Chem. 2001;276:33111–33120. [PubMed]
69. Sachdev S, Bruhn L, Sieber H, Pichler A, Melchior F, Grosschedl R. PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies. Genes Dev. 2001;15:3088–3103. [PubMed]
70. Takahashi Y, Toh-e A, Kikuchi Y. A novel factor required for the SUMO1/Smt3 conjugation of yeast septins. Gene. 2001;275:223–231. [PubMed]
71. Kahyo T, Nishida T, Yasuda H. Involvement of PIAS1 in the sumoylation of tumor suppressor p53. Mol Cell. 2001;8:713–718. [PubMed]
72. Johnson ES, Gupta AA. An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell. 2001;106:735–744. [PubMed]
73. Wu G, Xu G, Schulman BA, Jeffrey PD, Harper JW, Pavletich NP. Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase. Mol Cell. 2003;11:1445–1456. [PubMed]
74. Schulman BA, Carrano AC, Jeffrey PD, Bowen Z, Kinnucan ER, Finnin MS, Elledge SJ, Harper JW, Pagano M, Pavletich NP. Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex. Nature. 2000;408:381–386. [PubMed]
75. Min JH, Yang H, Ivan M, Gertler F, Kaelin WG, Pavletich NP. Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling. Science. 2002;296:1886–1889. [PubMed]
76. Hon WC, Wilson MI, Harlos K, Claridge TD, Schofield CJ, Pugh CW, Maxwell PH, Ratcliffe PJ, Stuart DI, Jones EY. Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL. Nature. 2002;417:975–978. [PubMed]
77. Fersht A. Enzyme Structure and Mechanism. W. H. Freeman; New York: 1984.
78. Gmachl M, Gieffers C, Podtelejnikov AV, Mann M, Peters JM. The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex. Proc Natl Acad Sci U S A. 2000;97:8973–8978. [PubMed]
79. Tang Z, Li B, Bharadwaj R, Zhu H, Ozkan E, Hakala K, Deisenhofer J, Yu H. APC2 Cullin protein and APC11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex. Mol Biol Cell. 2001;12:3839–3851. [PMC free article] [PubMed]
80. Seol JH, Feldman RM, Zachariae W, Shevchenko A, Correll CC, Lyapina S, Chi Y, Galova M, Claypool J, Sandmeyer S, Nasmyth K, Deshaies RJ. Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. Genes Dev. 1999;13:1614–1626. [PubMed]
81. Deffenbaugh AE, Scaglione KM, Zhang L, Moore JM, Buranda T, Sklar LA, Skowyra D. Release of ubiquitin-charged Cdc34-S - Ub from the RING domain is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Cell. 2003;114:611–622. [PubMed]
82. Siepmann TJ, Bohnsack RN, Tokgoz Z, Baboshina OV, Haas AL. Protein interactions within the N-end rule ubiquitin ligation pathway. J Biol Chem. 2003;278:9448–9457. [PubMed]
83. Wang M, Pickart CM. Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis. EMBO J. 2005;24:4324–4333. [PubMed]
84. Pichler A, Gast A, Seeler JS, Dejean A, Melchior F. The Nucleoporin RanBP2 Has SUMO1 E3 Ligase Activity. Cell. 2002;108:109–120. [PubMed]
85. Reverter D, Lima CD. Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex. Nature. 2005;435:687–692. [PMC free article] [PubMed]
86. Tatham MH, Kim S, Jaffray E, Song J, Chen Y, Hay RT. Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection. Nat Struct Mol Biol. 2005;12:67–74. [PubMed]
87. Haas AL, Rose IA. The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis. J Biol Chem. 1982;257:10329–10337. [PubMed]
88. Bossis G, Chmielarska K, Gartner U, Pichler A, Stieger, Melchior F. A fluorescence resonance energy transfer-based assay to study SUMO modification in solution. Methods Enzymol. 2005;398:20–32. [PubMed]
89. Lai Z, Ferry KV, Diamond MA, Wee KE, Kim YB, Ma J, Yang T, Benfield PA, Copeland RA, Auger KR. Human mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization. J Biol Chem. 2001;276:31357–31367. [PubMed]
90. Wilkinson KD. Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J. 1997;11:1245–1256. [PubMed]
91. Pickart CM, Rose IA. Ubiquitin carboxyl-terminal hydrolase acts on ubiquitin carboxyl-terminal amides. J Biol Chem. 1985;260:7903–7910. [PubMed]
92. Tobias JW, Varshavsky A. Cloning and functional analysis of the ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae. J Biol Chem. 1991;266:12021–12028. [PubMed]
93. Li SJ, Hochstrasser M. A new protease required for cell-cycle progression in yeast. Nature. 1999;398:246–251. [PubMed]
94. Gan-Erdene T, Nagamalleswari K, Yin L, Wu K, Pan ZQ, Wilkinson KD. Identification and characterization of DEN1, a deneddylase of the ULP family. J Biol Chem. 2003;278:28892–28900. [PubMed]
95. Wu K, Yamoah K, Dolios G, Gan-Erdene T, Tan P, Chen A, Lee CG, Wei N, Wilkinson KD, Wang R, Pan ZQ. DEN1 is a dual function protease capable of processing the C terminus of Nedd8 and deconjugating hyper-neddylated CUL1. J Biol Chem. 2003;278:28882–28891. [PubMed]
96. Mendoza HM, Shen LN, Botting C, Lewis A, Chen J, Ink B, Hay RT. NEDP1, a highly conserved cysteine protease that deNEDDylates Cullins. J Biol Chem. 2003;278:25637–25643. [PubMed]
97. Ambroggio XI, Rees DC, Deshaies RJ. JAMM: a metalloprotease-like zinc site in the proteasome and signalosome. PLoS Biol. 2004;2:E2. [PMC free article] [PubMed]
98. Balakirev MY, Tcherniuk SO, Jaquinod M, Chroboczek J. Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Rep. 2003;4:517–522. [PubMed]
99. Nanao MH, Tcherniuk SO, Chroboczek J, Dideberg O, Dessen A, Balakirev MY. Crystal structure of human otubain 2. EMBO Rep. 2004;5:783–788. [PubMed]
100. Reverter D, Wu K, Erdene TG, Pan ZQ, Wilkinson KD, Lima CD. Structure of a complex between Nedd8 and the Ulp/Senp protease family member Den1. J Mol Biol. 2005;345:141–151. [PubMed]
101. Shen LN, Liu H, Dong C, Xirodimas D, Naismith JH, Hay RT. Structural basis of NEDD8 ubiquitin discrimination by the deNEDDylating enzyme NEDP1. EMBO J. 2005;24:1341–1351. [PubMed]
102. Mossessova E, Lima CD. Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol Cell. 2000;5:865–876. [PubMed]
103. Johnston SC, Larsen CN, Cook WJ, Wilkinson KD, Hill CP. Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution. EMBO J. 1997;16:3787–3796. [PubMed]
104. Hu M, Li P, Li M, Li W, Yao T, Wu JW, Gu W, Cohen RE, Shi Y. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell. 2002;111:1041–1054. [PubMed]
105. McCullough J, Clague MJ, Urbe S. AMSH is an endosome-associated ubiquitin isopeptidase. J Cell Biol. 2004;166:487–492. [PMC free article] [PubMed]
106. Kovalenko A, Chable-Bessia C, Cantarella G, Israel A, Wallach D, Courtois G. The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination. Nature. 2003;424:801–805. [PubMed]
107. Brooks CL, Gu W. Dynamics in the p53-Mdm2 ubiquitination pathway. Cell Cycle. 2004;3:895–899. [PubMed]
108. Liu Y, Lashuel HA, Choi S, Xing X, Case A, Ni J, Yeh LA, Cuny GD, Stein RL, Lansbury PT. Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line. Chem Biol. 2003;10:837–846. [PubMed]
109. Desterro JM, Rodriguez MS, Hay RT. SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Mol Cell. 1998;2:233–239. [PubMed]
110. Xirodimas DP, Saville MK, Bourdon JC, Hay RT, Lane DP. Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity. Cell. 2004;118:83–97. [PubMed]
111. Shiio Y, Eisenman RN. Histone sumoylation is associated with transcriptional repression. Proc Natl Acad Sci U S A. 2003;100:13225–13230. [PubMed]
112. Girdwood D, Bumpass D, Vaughan OA, Thain A, Anderson LA, Snowden AW, Garcia-Wilson E, Perkins ND, Hay RT. P300 transcriptional repression is mediated by SUMO modification. Mol Cell. 2003;11:1043–1054. [PubMed]
113. Sun ZW, Allis CD. Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature. 2002;418:104–108. [PubMed]
114. Song J, Durrin LK, Wilkinson TA, Krontiris TG, Chen Y. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc Natl Acad Sci U S A. 2004;101:14373–14378. [PubMed]