Ubiquitylation provides a rapid alternative to control the activity of crucial cellular factors through the remodelling of a target protein. Diverse ubiquitin chains are recognized by domains with affinity for UBDs (ubiquitin-binding domains) present in receptor/effector proteins. Interestingly, some proteins contain more than one UBD and the preservation of this structure in many species suggests an evolutionary advantage for this topology. Here, we review some typical proteins that naturally contain more than one UBD and emphasize how such structures contribute to the mechanism they mediate. Characteristics such as higher affinities for polyubiquitin chains and chain-linkage preferences can be replicated by the TUBEs (tandem ubiquitin-binding entities). Furthermore, TUBEs show two additional properties: protection of ubiquitylated substrates from deubiquitylating enzymes and interference with the action of the proteasome. Consequently, TUBEs behave as ‘ubiquitin traps’ that efficiently capture endogenous ubiquitylated proteins. Interpretations and hypothetical models proposed by different groups to understand the synchronous action of multiple UBDs are discussed herein.
Ubiquitylation determines the destiny of a protein by remodelling its folding and rapidly changing its properties. This amazing plasticity is partly due to the capacity of ubiquitin to form monomers (mono-ubiquitylation) and polymers (polyubiquitylation) using any of the seven lysine residues of ubiquitin (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63). While the precise mechanisms ensuring linkage-specific polyubiquitylation are still unknown , some generic functions have been attributed to Lys48 (targeting proteins for proteolysis) and Lys63 (involved in DNA repair and signal transduction) poly-linkages . Moreover, ubiquitylation complexity increases with the possible formation of heterologous chains anchored at different lysine acceptors of the same protein substrate , and with the recently discovered hybrid SUMO (small ubiquitin-related modifier) -2/3-ubiquitin chains [4,5].
Ubiquitylation is a highly dynamic reversible process driven by the action of specific DUBs (deubiquitylating enzymes), which remove ubiquitin moieties from modified substrates to regulate protein stability and function. DUBs show very heterologous structures and are involved in diverse processes. In addition to the catalytic site, all DUBs bind ubiquitin using one or more UBDs (ubiquitin-binding domains), which recognize ubiquitin from the other ubiquitin-like modifiers and also contribute to discriminate between different ubiquitin chain linkages . UBDs have a more extended role in the co-ordination of ubiquitin-regulated processes, acting as ubiquitin receptors to allow the connection with effector functions. At least 20 families of UBDs have been reported, including UBA (ubiquitin-associated) domain, and UIM (ubiquitin-interacting motif) among others [3,7,8]. Although UBDs show relatively low sequence conservation, the largest class, including UBA and UIM domains, contains an α-helical arrangement that interacts through a common region containing a hydrophobic patch consisting of Leu8, Ile44 and Val70 [7,9]. UBA domains have been classified in four groups according to their ubiquitin-chain-binding preferences. Classes 1 and 2 show a preferential binding to Lys48 and Lys63 ubiquitin chains respectively, class 3 do not bind any polyubiquitin chains and class 4 do not present any linkage preference . The affinity of UBA domains for mono-ubiquitin is in general quite low (a few hundred micromolar), whereas affinity for polyubiquitin chains is increased up to 70 times higher (low micromolar range), depending on the nature of the domain . Apart from the UBA domains, the UIMs are the most widespread UBDs. This motif is composed of ~20 amino acid residues and was originally identified as a polyubiquitin-binding site in the S5a subunit (called RPN10) of the 26S proteasome. IUM domains are often found in proteins that function in the pathways of endocytosis and vacuolar sorting [11,12]. The structure of the UIM domain consists of a single amphipathic α-helix, capped at the N-terminus by a cluster of negatively charged residues. The UIM binds to mono-ubiquitin with a Kd value of 0.1–1 mM . While the presence of a single UBD appears to be sufficient for the recognition of ubiquitylated substrates, the presence of multiple UBDs in a single ubiquitin receptor protein argues in favour of multiple synchronous or sequential events required to co-ordinate a function (Figure 1). Protein receptors may contain homologous or heterologous UBDs providing distinctive properties and roles.
Domain structure of proteins with multiple UBDs
Proteins containing multiple UBA domains
Polyubiquitylated proteins bind directly to the 26S proteasome but also through adaptor proteins that display UBL (ubiquitin-like) domain–UBA domains. The hHR23A (human homologue radiation-sensitive mutant 23A) protein contains two UBA domains, one internal (UBA1) and one in the C-terminus (UBA2) (Figure 1). Both domains interact weakly with mono-ubiquitin and strongly with polyubiquitin [10,14]. While the UBA2 has been classified in category 1 given its preference for Lys48 linked chains, the UBA1 is considered to be in category 2 as it binds Lys63, Lys48 and Lys29/6 tetra-ubiquitin chains. The structure of the UBA2 domain supports a model where the Lys48-linked di-ubiquitin wraps around the UBA domain, making an extensive contact. This mode of interaction might explain the observed increased affinity of the UBA2 for tetra-ubiquitin chains. Also, it offers a plausible explanation for the Lys48-chain preference versus Lys63 tetra-ubiquitin, which adopts an extended linear conformation in solution . Indeed, Lys48 chains form globular structures where the ubiquitin hydrophobic surface patch is hidden due to intramolecular ubiquitin interactions [15,16]. As suggested for other UBL–UBA proteins, hHR23A has been implicated in the presentation of ubiquitylated proteins to the 26S proteasome via its interaction with the UBL domain (Figure 2A). Both UBA-hRad23A domains interact with its UBL domain to regulate interactions of polyubiquitin chains with the proteasome [14,17]. However, when isolated UBA domains are used in vitro, they inhibit the action of the proteasome [18,19]. UBA domains were found to be essential for homodimerization of Rad23 and heterodimerization between Rad23 and the UBL–UBA protein Ddi1. Furthermore, Rad23 and Ddi1 bind ubiquitin but the dimerization of Rad23 blocks ubiquitin binding, thus suggesting a possible mechanism for regulating Rad23 and Ddi1 function .
Hypothetical models of regulation of proteins encoding multiple UBDs
The human NUB-1 [NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) ultimate buster-1] and its splicing variant NUB-1L are negative regulators of the ubiquitin-like molecule NEDD8 conjugation system, which brings NEDD8 and its targets to the proteasome. Both Human NUB-1 and NUB-1L contain one UBL and two or three UBA domains respectively that surprisingly do not bind to ubiquitin . Therefore they can be included in group 3 of UBA domains . NUB-1L also interacts with the ubiquitin-like modifier FAT10 which is induced by IFNγ (interferon γ) and TNFα (tumour necrosis factor α) . FAT10 contains two UBL domains that target long-lived proteins for rapid proteasomal degradation. It has been reported previously that NUB-1L associates with FAT10 and its substrates to strongly down-regulate their expression by accelerating their degradation by the proteasome . This process does not depend on the three UBA domains, but rather on the NUB-1L-UBL domain, supporting its role as a facilitator of proteasomal degradation .
Proteins containing multiple UIMs
UIMs are found in a number of proteins involved in endocytosis and protein trafficking that can bind preferentially mono-ubiquitin but also polyubiquitin chains . UIMs have also been shown to bind to ubiquitin-like domains, e.g. the UBL of hHR23B, providing a mechanism of regulation for proteins displaying UBL–UBA domains . UIMs are found often in tandem [RAP80 (receptor-associated protein 80) and S5a] or triplet arrays [USP (ubiquitin-specific peptidase) 37 and ataxin-3], in proteins involved in ubiquitylation and ubiquitin metabolism (Figure 1). The proteasomal subunit S5a uses its multiple UIM domains to recognize Lys48-modified proteins for proteasomal degradation . Other proteins such as ataxin-3 and RAP80 use multiple UIM domains to recognize ubiquitin chains of different linkages [28–30]. Ataxin-3 binds both Lys48- and Lys63-linked ubiquitin chains, yet its DUB activity preferentially cleaves Lys63-linked ubiquitin chains in vitro , suggesting that the UIMs present in ataxin-3 are required both for the cleavage and recognition of ubiquitin chains . RAP80 contains a double UIM that recognizes Lys63-linked polyubiquitin chains. This protein is a key player in the recruitment of repair proteins to DNA damage sites by recognizing specifically Lys63-linked polyubiquitin chains . Sato et al.  recently published the structural basis for the recognition of the RAP80–UIM1–UIM2 motifs in complex with Lys63 di-ubiquitin. Such recognition implicates the α-helical inter-UIM region that selectively binds Lys63-linked di-ubiquitin [31–33]. Taking into account that the affinity between UIMs and mono-ubiquitin is low, the observed robust interaction between RAP80 and Lys63-linked polyubiquitin substrates is probably achieved by co-operative binding between multiple UIMs and ubiquitin chains interacting through an avidity-based mechanism, where binding by the first of multiple UIMs to one ubiquitin would position the second UIM favourably for interaction with a nearby ubiquitin in the chain .
Proteins containing heterologous UBDs
USP5, also known as isopeptidase T, is a DUB mainly responsible for the disassembly of unanchored polyubiquitin chains in vivo [34–36]. The elimination of free polyubiquitin chains reduces the competition for the binding of polyubiquitylated substrates to many receptors involved in multiple cellular events. In vitro, USP5 acts on Lys48, Lys63, Lys29/6 and linear ubiquitin chains [10,35]. The cleavage of Lys48 chains occurs on the ubiquitin that contains the free C-terminus of the unanchored chains . USP5 contains four UBDs: a ZnF (zinc finger) UBP (ubiquitin-specific processing protease) that binds to the proximal ubiquitin, the UBP domains that form the active site, and two UBA domains involved in the binding of at least Lys48 and linear polyubiquitin chains (Figure 1). While the binding to polyubiquitin chains is co-ordinated by the four UBDs, mutation studies indicate that the UBA1 and UBA2 of USP5 interact with the third and fourth ubiquitin of Lys48 and linear polyubiquitin chains  (Figure 2B). The recognition of polyubiquitin by the several UBDs present in USP5 might increase the efficiency of the processing and elimination of unanchored ubiquitin polymers .
USP25m is the muscle isoform of the DUB USP25. In silico analysis predicted three UBDs at the N-terminus of all USP25 isoforms: one UBA and two UIMs (Figure 1) . The UBDs of USP25m are not strictly required for the recognition and rescue of one of its substrates, MyBPC1, from proteasome degradation, but they seem to modulate the enzyme activity, by either direct enzyme regulation, altering access to the substrate or shifting the preferential binding to different ubiquitin chains, as occurs in other enzymes and processes. Furthermore, the UBDs favour the mono-ubiquitylation of USP25m at the preferential site Lys99 located at UIM1, a residue that was previously shown to be a target for SUMO . SUMOylation in Lys99 inhibited USP25 activity, as it did the mutation K99R, which abrogated the two modifications. Interestingly, USP25 is able to autodeubiquitylate in a possible loop of autoregulation. Overall, these results suggest a model where USP25m is activated by ubiquitylation and inhibited by SUMOylation on the same lysine residue , and these modifications depend on the complex interplay of the UBDs and an N-terminal SIM (SUMO-interacting motif) (Figures 1 and 2C). Almost simultaneously, the DUB activity of ataxin-3 was shown to be positively regulated by ubiquitylation. Although the 3 UIMs are dispensable for the ubiquitin dependent activation of ataxin-3, they do confer preference for Lys63 linkages . These convergent forms for the regulation of DUBs suggest that preserved mechanisms control the cleavage of distinct polyubiquitin chains and/or provide substrate specificity.
Artificial proteins containing multiple UBDs
Intrigued by the many examples of naturally existing proteins containing more than one UBD and considering that multiplicity of domains with affinity for ubiquitin could exacerbate/exponentially accentuate some of their characteristics, we were prompted to explore whether the co-operative ubiquitin-binding effect observed with some proteins containing more than one UBD could be artificially reproduced. Based on the UBA of hHR23A (UBA1) and ubiquilin-1, TUBEs (tandem ubiquitin-binding entities) were developed to purify ubiquitylated proteins . The artificial disposition of several UBA domains separated with a flexible linker, does not appear to affect their capacity to specifically recognize ubiquitin. Using SPR (surface plasmon resonance), TUBEs were found to display a remarkable increase in affinity for polyubiquitin chains going from the low-micromolar to the low-nanomolar range. TUBE-ubiquilin-1 preserved a slight preference for Lys63 versus Lys48 chains observed with the single domain while the TUBE-hHR23A showed similar affinity for both chain types . As a consequence of the 103-fold higher affinity for polyubiquitin chains compared with a single UBD domain, TUBEs are a very efficient tool for purification of ubiquitylated proteins from cell extracts, tissues and organs under native conditions (Figure 3A). Interestingly, TUBEs show two other characteristics that can be used independently in ex vivo and in vitro assays: TUBEs protect ubiquitin-conjugated proteins from both proteasomal degradation (Figure 3B) and the action of DUBs present in cell extracts, at least to the same extent as the widely used proteasome and cysteine protease inhibitors NEM (N-ethylmaleimide) and IAA (iodoacetamide) (Figure 3C) . Although more results are required to understand all TUBEs properties, the amino acid composition of the UBA domains is likely to determine the preferences for different ubiquitin chain linkages and confer diverse solubility properties, according to the hydrophobic nature of the domain. Further, intra- or inter-molecular interactions might modulate the capacity of some TUBEs to capture ubiquitylated proteins. Even if the UBA domains selected when developing the current TUBE prototypes behave as pan-ubiquitin-linkage reagents, they inherently possess chain preferences and thus, render slightly different results. This becomes evident when two different TUBEs (each of them containing homologous UBA domains) are simultaneously used to capture ubiquitylated proteins . Indeed, the analysis of the captured material will provide information about known and unknown ubiquitylated proteins, but also about proteins interacting with these modified cell factors referred to as ubiquitin-interactomes (Figure 2D). The interactome of ubiquitin-modified proteins remains largely unknown and such knowledge may lead to the identification of new targets for drug development to treat disorders such as cancer and neurodegeneration.
Characteristics of artificial proteins encoding multiple UBA domains (TUBEs)
Multiple sites for ubiquitin interaction appear to guarantee the efficacy of ubiquitin recognition in multiple processes. Co-ordination and co-operativity during the recognition of polyubiquitin chains is a plausible rationale for the presence of UBD arrays in the same protein. These characteristics must be crucial for critical processes, such as proteasomal degradation or the inherent selectivity of DUBs. Some of these traits can be mimicked by artificial proteins displaying multiple UBDs, with a remarkable increase in affinity for polyubiquitin chains compared with single UBA domains. Consequently, TUBEs are efficient tools for purification of ubiquitylated proteins from cell extracts under native conditions. Furthermore, TUBEs preserve two useful advantages of the UBA domains: they protect ubiquitin-conjugated proteins from both proteasomal degradation and the DUB activities present in cell extracts to a similar extent as widely used proteasome and cysteine protease inhibitors. Capturing endogenously ubiquitylated proteins with such efficiency is an attribute not provided by any other tool or technique used until now.
Ubiquitin–Proteasome System, Dynamics and Targeting: 4th Intracellular Proteolysis Meeting, a Biochemical Society Focused Meeting held at Institut d'Estudis Catalans, Casa de Convalescència, Barcelona, Spain, 27–29 May 2009. Organized and Edited by Bernat Crosas (Institute of Molecular Biology of Barcelona, Spain), Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (University of Barcelona, Spain), Manuel Rodríguez (CIC bioGUNE, Derio, Spain) and Timothy Thomson (Institute of Molecular Biology of Barcelona, Spain)
human homologue radiation-sensitive mutant 23A
NEDD8 (neural precursor cell expressed, developmentally down regulated 8) ultimate buster-1
receptor-associated protein 80
small ubiquitin-related modifier
tandem ubiquitin-binding entity
We thank Valerie Lang and Gemma Marfany for the critical reading of the manuscript.
This work was funded by the Ramón y Cajal Programme, Ministerio de Educación y Ciencia (Spain) [grant number BFU 2005-04091], the Fondo de Investigaciones Sanitarias, CIBERhed, the Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Etortek Research Programmes 2008/2010) and by the Innovation Technology Department of the Bizkaia Country.