Ubiquitin classes

Examples of such systems include the reactions of kinases, phosphatases, hydroxylases, acetylases, ubiquitin transferases, and many other enzyme classes that represent attractive targets for drug discovery. There are several mechanisms by which an enzyme can catalyze these types of reactions, and the details of the mechanism are important in determining the best approach to designing activity assays for the enzyme and for proper evaluation of inhibitors that are identified through those activity assays. 

CoscoY, L., D. J. Sanchez, and D. Ganem, a novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition. / Cell Biol, 2001, 155(7), 1265-73. 

Bartee, E., et ah, Downregulation of major histocompatibility complex class I by human ubiquitin ligases related to viral immune evasion proteins. J Virol, 2004, 78(3), 1109-20. 

Cook, W. J., Jeffrey, L. C., Xu, Y., and Chau, V. Tertiary structures of class 1 ubiquitin-conjugating enzymes are highly conserved crystal structure of yeast Ubc4. Biochemistry 1993, 32, 13809-17. 

Miura, T., Klaus, W., Gsell, B., Miyamoto, C., and Senn, H. Characterization of the binding interface between ubiquitin and class 1 human ubiquitin-conjugating enzyme 2b by multidimensional heteronudear NMR spectroscopy in solution./. Mol. Biol. 1999, 290, 213-28. 

TSGlOl may be the prototype of a class of dominant negative ubiquitin regulators. Nature Genet. 1997, 16,... 

The first class of DUBs discovered, the ubiquitin C-terminal Aydrolases (UCHs), is a relatively small class vith only four members in humans and one in budding yeast. UCHs are cysteine proteases related to the papain family of cysteine proteases. Most UCHs consist entirely of a catalytic core that has a molecular mass of about 25 kDa, although Bapl and UCH37 have C-terminal extensions [21, 22], All UCHs have a highly conserved catalytic triad consisting of the active-site cysteine, histidine, and aspartate residues that are absolutely required for function [23]. 

Fig. 8.2. Substrate binding by DUBs reveaied by X-ray crystai structures, in the ribbon diagrams, the DUB is represented in white and the substrate in coior. The ubiquitin (yeiiow or green) or SUMO (red) substrates are shown in the same orientation to highlight the similarity of substrate binding by different DUB classes. (A) Ubiquitin (yellow) bound to YUHl. (B)...

The ubiquitin specific processing proteases (referred to as UBPs in yeast and USPs in human and mouse) were the second class of DUBs discovered. Catalytically, the UBPs are very similar to the UCHs in that they also utilize the catalytic triad of an active-site cysteine and a conserved histidine and aspartate. The UBP catalytic core... 

The ubiquitin-7ike specific proteases (ULPs) are a third class of DUB first thought to act only on SUMO-related ubiquitin-like proteins. There are two yeast ULPs and seven human ULPs (also called sentrin specific proteases, or SENPs). Further analysis determined that ULPs have little or no activity on ubiquitin substrates, but one (SENPS) acts on NeddS [26, 37, 38]. Despite acting on non-ubiquitin substrates,... 

A class of DUBs only identified since 2002 is the OTU (ovarian tumor protein) DUB class. The OTU domain was originally identified in an ovarian tumor protein from Drosophila mdanogaster, and over 100 proteins from organisms ranging from bacteria to humans are annotated as having an OTU domain. The members of this protein superfamily were annotated as cysteine proteases, but no specific function had been demonstrated for any of these proteins. The first hint of a role for OTU proteins in the ubiquitin pathway was afforded by the observation that an OTU-domain-containing protein, HSPC263, reacted with ubiquitin vinyl sulfone (an active-site-directed irreversible inhibitor of DUBs) [41]. 

Despite the initial indication that Rpnl0/S5a was the polyubiquitin-chain-binding subunit of the 26S proteasome, subsequent studies showed that it cannot be the principal recognition element for this process. First, disruption of the Rpnl0/S5a gene in yeast is not lethal and inhibits the degradation of only a sub-class of ubiquitinated proteins [39]. Likewise, RNA interference-inhibited ex-... 

While the functional analogy between ThiS, MoaD, and ubiquitin-like modifiers is widely accepted, the two protein classes are frequently described as unrelated sometimes even a convergent evolution to the energetically favorable ubiquitin fold is discussed. Despite these claims, there is a statistically significant sequence... 

FI/F2/F3, triad of RING-finger-like domains F, F-box domain USP, deubiquitinase catalytic domain OTU, a particular class of cystein protease domains RVP, retroviral protease domain DBA, ubiquitin-associated domain Pkinase, protein kinase catalytic domain. 

Finally, it should be mentioned that there are a number of protein domains that have some structural resemblance to ubiquitin, although a sequence similarity cannot be established - not even by the most sophisticated methods available today. It cannot be excluded that there are true instances of convergent evolution among these cases. However, it appears more likely that these proteins and domains represent distant members of the ubiquitin superfamily, which have undergone a fundamental change of function and no longer need to conserve sequence positions that are considered hallmarks of ubiquitin-like molecules. In particular three domain classes should be mentioned in this context. The PERM domain (4.1, ezrin,... 

In addition, there are a number of borderline cases, whose sequence relationship to ubiquitin is hard to establish but most probably is real, as these proteins perform a similar function. One well-known example is the UBX domain [44, 45], which seems to replace an internal ubiquitin domain in a certain class of adapter proteins (see Section 12.5.2). Other examples are the autophagy proteins Apg8 and Apgl2 [23, 24] which act as ubiquitin-like modifiers. 

In this respect, the CUE domain is not a isolated case. There are a number of other domain families, each of them only defined in the bioinformatical sense, that have significant matches within established UBA or CUE domain regions. Based on this similarity and on secondary-structure predictions, it can be expected that all of those domain types assume the typical UBA-like three-helix bundle fold. However, it is not clear if all of those domains also bind to ubiquitin, or if they have evolved to different binding properties. Many of the UBA-like domain classes are unpublished. Nevertheless, they should be briefly discussed here, as they are a logical extension of the UBA/CUE paradigm. 

The GAT domain (GGA and Toml) has recently joined the ranks of ubiquitin-binding domains [91]. As the name implies, this domain is found in the GGA-and Toml-like proteins, two regulator classes of clathrin-mediated vesicular traffic. All proteins harboring the GAT domain also contain an N-terminal VHS domain, which is named after the Vps27, Hrs, and STAM proteins. Interestingly, these latter proteins are known to contain a ubiquitin-binding UIM motif, which appears to be replaced by the GAT domain in the GGA and Toml-like proteins (Figure 12.6). 

Initially, the role of the GAT domain in GGA proteins was seen in the binding of small GTPases of the Arf family, a critical step in the recruitment of clathrin to the TGN membrane [92]. However, the GAT domains of the Toml-like family do not bind to Arf. Recently, GAT domains of both protein classes were found to bind to ubiquitin and it was possible to separate the two binding sites to different subdomains of the GAT domain [91]. A number of X-ray structures of GAT domains are available [93-95], presenting the domain as an elongated three-helix bundle. Unlike the UBA-like structures, the GAT helices are almost parallel and considerably longer. As a prominent feature, the N-terminal helix is much longer than the others this N-terminal extension contains the Arf interaction site and is not conserved in the Toml-family [93]. 

Obviously, not all proteins known to interact with ubiquitin or ubiquitin-like domains contain one of the professional ubiquitin-interaction domains. RptS and Rpnl, two subunits of the proteasome that bind to ubiquitin and UbLs, respectively, do not belong to any of the classes described above. Most probably, a large number of uncharacterized proteins with high affinity and specificity for ubiquitin are still waiting to be discovered. The bioinformatical tools described in the early sections of this chapter will be instrumental for this task. 

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