Ub (ubiquitin) and Ubls (Ub-like molecules) are peptide modifiers that change the fate and function of their substrates. A plethora of enzyme activities and protein cofactors are required for either the conjugation (mainly E3 ligases) or deconjugation of Ub and Ubls. Most of the data have been gathered on describing individual enzymes and their partners, but an increasing number of reports point to the formation of multisubunit complexes regulated by cross-talk between Ub and Ubl systems and which contain opposing conjugation/deconjugation activities. This minireview focuses on these latest reports and proposes that these complexes, which are able to recruit transient partners, shift cofactors and integrate different signalling stimuli, are a common strategy to regulate highly dynamic processes, in a switch-on/switch-off type of mechanism, thus responding promptly to cellular requirements.

The background

Ub (ubiquitin) and Ubl (Ub-like molecule) conjugation and deconjugation

Ub and Ubls are molecular tags used by eukaryotic cells to determine protein fate and regulate protein function. Ub and Ubls are relatively small peptides that are post-translationally conjugated to protein substrates, thus providing new protein–protein interaction interfaces and changing protein recognition and binding affinity. Ub (and also Ubls) can be conjugated as single moieties or as poly-Ub chains, and the final number of conjugated Ubs, as well as the lysine used for the Ub-chain growth, are the determinant for both the fate and function of the modified protein.

A long poly-Ub chain usually targets the substrate for proteasome degradation, but addition of Ub or Ubl molecules as single moieties can have disparate effects: from shuttling a substrate to a particular cellular compartment, to activating or inactivating an enzyme, to regulating interactions with proteins involved in signalling pathways. Therefore, and albeit initially described in the context of intracellular protein degradation, the relevance of Ub and Ubl conjugation in the regulation of protein activity and fate is nowadays unquestionable [1,2].

Ubiquitination and deubiquitination have been often compared with other post-translational modifications, e.g. phosphorylation and glycosylation, and also as one of such, eukaryotic cells deploy a battery of enzymes to either promote or reverse Ub (or Ubl) conjugation depending on cellular requirements. Therefore the state of a particular substrate depends on the balance between the ubiquitinating and the deubiquitinating activities acting on it [3]. The conjugation enzymes, the Ub ligases, work hierarchically on substrates: the E1-activating enzyme passes an activated Ub to an E2-Ub conjugating enzyme, which is then able to bind an E3 Ub protein ligase. E3s bring together the substrate to the Ub-loaded E2 and allow the Ub conjugation to the substrate and, hence, E3 provides substrate specificity. Not only the conjugation of the first Ub, but also the growth of the poly-Ub chain depends on the successive interaction of E2 and E3 ligases, usually in versatile heteromeric complexes with the recruitment of distinct cofactors [4]. Recently, and for mono-ubiquitination, a mechanism independent of E3 ligases, but strictly dependent on E2 ligases has also been proposed [5]. Table 1 summarizes the current classification of Ub E3 ligases in families and types, according to structural domains relationships and similarity on the mechanisms for Ub conjugation (reviewed in [610]).

Table 1
Human E3 ligase families, nomenclature and examples (after [610])
Families Abbreviation Subfamily Examples 
Homologous to E6-associated protein C-terminus HECT HERC (HECT and RCC1-likeHERC5 
  Nedd4/Nedd4-like Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4), Smurf2 
  SI-HECT (single HECTE6-AP 
Cullin-RING E3 Ub ligases RING SCF (Skp1/cullin/F-boxRbx1, Cul1, Skp1, F-box proteins 
  APC (anaphase-promoting complex/cyclosomeAPC2, APC11 
  CBC (Cul2Elongin BElongin CRbx1, Cul2, Elongin, SOCS (suppressor of cytokine signalling)-box 
  SPRF Cbl, Mdm2 
UFD2 homology (U-box) proteins U-box  CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein], UFD2, CYC4, PRP19 
Plant homeodomain PHD  MEKK1 {MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] kinase 1} 
Families Abbreviation Subfamily Examples 
Homologous to E6-associated protein C-terminus HECT HERC (HECT and RCC1-likeHERC5 
  Nedd4/Nedd4-like Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4), Smurf2 
  SI-HECT (single HECTE6-AP 
Cullin-RING E3 Ub ligases RING SCF (Skp1/cullin/F-boxRbx1, Cul1, Skp1, F-box proteins 
  APC (anaphase-promoting complex/cyclosomeAPC2, APC11 
  CBC (Cul2Elongin BElongin CRbx1, Cul2, Elongin, SOCS (suppressor of cytokine signalling)-box 
  SPRF Cbl, Mdm2 
UFD2 homology (U-box) proteins U-box  CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein], UFD2, CYC4, PRP19 
Plant homeodomain PHD  MEKK1 {MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] kinase 1} 

Ub deconjugation or Ub editing is performed by DUBs (deubiquitinating enzymes). So far there are five different DUB families, with distinct traits and catalytic domains (reviewed in [1113], see Table 2 for a summary), but the most extended so far is the UBP [Ub-specific protease; also named USP (human Ub-specific protease) after human enzymes and genes] family. While there are more than 500 different Ub ligases, DUBs amount to approx. 100 different genes in the human genome. Most of the information on the UPS (Ub/proteasome system) has been gathered on the activity of Ub E3 ligases, on their specificity and regulation. In contrast, data on the activity, specificity and regulation of DUBs are relatively scanty, although lately it is becoming one of the burgeoning fields in the UPS.

Table 2
Human deubiquitinating enzyme families and nomenclature (after [1113])
Families Abbreviation Type of catalysis Examples 
Ubiquitin C-terminal hydrolases UCH Cysteine protease UCH-L1 
Ubiquitin-specific proteases USP (UBP in non-human) Cysteine protease HAUSP (herpesvirus-associated ubiquitin- specific protease), UBPY, USP28, USP44 
Ovarian tumour proteins OTU Cysteine protease A20, Cezanne 
Machado–Joseph disease domain proteins MJD Cysteine protease Ataxin-3 
JAB1 (Jun activation domain-binding protein 1), MPN, MOV34 metalloproteases JAMM Metalloprotease Rpn11, AMSH, CSN5 
Families Abbreviation Type of catalysis Examples 
Ubiquitin C-terminal hydrolases UCH Cysteine protease UCH-L1 
Ubiquitin-specific proteases USP (UBP in non-human) Cysteine protease HAUSP (herpesvirus-associated ubiquitin- specific protease), UBPY, USP28, USP44 
Ovarian tumour proteins OTU Cysteine protease A20, Cezanne 
Machado–Joseph disease domain proteins MJD Cysteine protease Ataxin-3 
JAB1 (Jun activation domain-binding protein 1), MPN, MOV34 metalloproteases JAMM Metalloprotease Rpn11, AMSH, CSN5 

The effects of Ub and Ubl are mediated by Ub receptors: the UBDs (Ub-binding domains)

Most cellular effects of Ub and Ubls are mediated by proteins that contain specific UBDs. Several UBDs that recognize poly- or mono-Ub and Ubls have been identified, and the family of UBDs has been increasing rapidly during the past few years (reviewed in [14]). UBDs, as single domains or in arrays (belonging to the same or to different families), are found in all proteins that interact or are involved in the Ub pathway, from E2 and E3 ligases to DUBs, to many protein adaptors, cofactors, anchoring proteins and, in general, in Ub or Ubl effectors. UBDs are quickly becoming more than just a recognition interface: in many proteins, UBDs recognize intramolecular mono-Ub modifications in cis and are the means to regulate the balance between active or inactive proteins, or preventing the recognition of Ubs and Ubls in trans [15].

The hypothesis

E3 ligases and DUBs are dynamic components of multimeric platform complexes for protein modification

Many Ub and Ubl E3 ligases form part of multisubunit heteromeric complexes that include E2 ligases, but also adaptor proteins that bring together the corresponding protein substrates, the E2 and E3 activity subunits. Some of them also contain anchor proteins to bind the complex to particular cellular compartments [e.g. ER (endoplasmic reticulum), the NPC (nuclear pore complex), endosomal vesicles and microtubules], as well as regulatory proteins, which by different means, e.g. phosphorylation, ubiquitination, SUMOylation, neddylation etc. cross-talk (reviewed in [16]), regulate the activation and the tempo of the distinct enzymatic activities. Many of the interactions among the proteins of the complex are mediated by UBDs, contributing in different numbers, shapes and combinations. Therefore these complexes could be considered highly dynamic multifactorial platforms for protein modification, which not only integrate different cellular stimuli, but also, by recruiting distinct partners, control multiple aspects of cell cycle, transcription, signal transduction, DNA repair and development.

Notwithstanding that most information had been gathered on ubiquitinating ligases, their cofactors and their ability to form complexes, this minireview aims instead at highlighting some of the latest data that report reversible activities, namely conjugation and deconjugation, acting together in the same complex, hence: (i) focusing on the versatility of these multisubunit complexes; (ii) emphasizing that most Ub and Ubl modifications are most probably regulated in these types of complex; and (iii) proposing that not only E2 and E3 Ub or Ubl ligases are to be found in these complexes, but that the enzymes responsible for the reverse activities, i.e. DUBs, deSUMOylating and deneddylating enzymes, are also the main components that are dynamically recruited to allow an accurate and rapid response to the cellular requirements, often in a switch-on/switch-off effect (Figure 1).

Ub conjugation and deconjugation

Figure 1
Ub conjugation and deconjugation

(A) Multiple types of components for Ub- and Ubl-conjugation/deconjugation processes. (B) The regulation of Ub conjugation and deconjugation is proposed to be a dynamic balance between different recruited partners and activities, particularly in switch-on/switch-off processes.

Figure 1
Ub conjugation and deconjugation

(A) Multiple types of components for Ub- and Ubl-conjugation/deconjugation processes. (B) The regulation of Ub conjugation and deconjugation is proposed to be a dynamic balance between different recruited partners and activities, particularly in switch-on/switch-off processes.

Some data

DUB control of E3 ligases

While viral E3 RING (really interesting new gene) ligases appear to be stable, cellular E3 ligases are strictly controlled by an accurate balance between ubiquitination and deubiquitination. Indeed, most E3 RING ligases are capable of autoubiquitination, providing a self-regulatory mechanism of its half-life and activity by targeting themselves to degradation by the proteasome ([17,18] and references cited therein). DUBs acting on these Ub-conjugated ligases provide the counterpoint for regulation. For some E3 ligases, as recently reported, the regulation of the activity can be compartment-specific by recruitment of two different DUBs: USP9X for the rescue of the cytosolic E3 and USP7 for the nuclear E3 [18]. Indeed, these DUBs also play other cellular roles and are able to rescue other relevant proteins, most probably when recruited in other contexts: USP7 deubiquitinates FOXO4 (forkhead box O4) [19], as well as Mdm2 (murine double minute 2; another E3 ligase) and p53 (the substrate of Mdm2) [20], in a complex and fascinating feedback cross-talk, whereas USP9X is required for deubiquitinating critical cell adhesion as well as synaptic proteins [21].

ERAD (ER-associated degradation) and the deubiquitination prior to dislocation

ERAD is the primary mechanism of protein quality control within the secretory pathway. Misfolded or unassembled proteins in the ER are specifically recognized and targeted for proteasome degradation by distinct complexes containing several E2 and E3 ligases, anchoring proteins and substrate-recognizing factors, as well as translocation (also named dislocation) effectors for the subsequent shuttling of polyubiquitinated substrates through the ER membrane, and delivery to the cytosolic proteasomes [22,23]. Systematic approaches in yeast have allowed the classification of ERAD into distinct pathways (which share some subunits and enzymes), depending on whether the protein is misfolded at the luminal, transmembrane or cytosolic domains [24]. Most reports focused on the characterization of the E3 ligases and subunits of the complex; however, recent reports remark that a deubiquitinating enzyme, ataxin-3, binds to subunits of the ERAD complex and its activity is a requirement to proceed with the dislocation of misfolded proteins. Indeed, inhibition of this DUB activity causes accumulation of poly-Ub misfolded proteins in the ER, blocking ERAD effectively [25,26]. This is a promising pathway that merits further exploration, as arguably, other DUBs might be required for control editing or in several of the least-known ERAD mechanisms: (i) transport or dislocation through the ER membrane; (ii) requirement – or not – to unfold some of the targeted proteins in order to dislocate; and recognition, transport and delivery of the targeted proteins to the proteasome once they reach the cytosolic face of the ER.

SUMO (small Ub-related modifier) wrestling at the gate to the nucleus: the NPC

Reversibility of conjugation is not confined to Ub modification. One of the best-characterized Ubls, SUMO, is a relevant peptide in nuclear physiology: from nucleocytoplasmic transport, control of cell division, DNA repair, DNA replication, DNA transcription and mRNA quality control [27]. As it happens with Ub, not only SUMO ligases [28], but also deSUMOylating enzymes are relevant to the processes controlled by SUMO [29]. Over the past few years, many enzymes of the SUMO cycle have been localized at the NPCs. NPCs, formed by several arrayed interacting nucleoporin subunits, are the gatekeepers of the nuclear membrane, responsible for the traffic control between the nucleus and cytosol. It is well documented that in vertebrates at least, SUMO ligases and accompanying machinery are located at the cytosolic face of the NPC, but it is now clear that SUMO proteases anchored in the inner side of the NPC is an evolutionarily conserved feature. Both SUMO ligases and proteases are bound, by either direct or tethered interaction, to the large NPCs, thus becoming an even larger complex that could recruit these modifying activities depending on the cellular state [30]. An example of the close interplay of these two opposing activities in the same pathway comes from yeast mutants: disturbance of either SUMOylation or deSUMOylation enzymes of the NPCs blocks the nuclear import of proteins containing the classical nuclear localization sequence, as the recycling of the protein adaptor Kap60, which recognizes that sequence is impaired [31]. In addition, other yeast mutants of the NPC result in a profound disturbance of the SUMO pathway, with impairment of DNA replication, DNA double-strand break repair and even mRNA quality control surveillance. Overall, pointing to a close relationship between the SUMO-regulated processes (and thus the SUMO-conjugation/deconjugation enzymes) and the NPCs.

E3 conjugation and DUB edition: endosomal sorting complexes, and the mitotic spindle connection

Proteins tagged to lysosomes (such as the epidermal growth receptor) are tagged by Ub. These proteins evade proteasomal degradation because they are mono- or di-ubiquitinated (using a Lys-63 chain) instead of polyubiquitinated (using a Lys-48 chain). They are thus incorporated into ESCRT (endosomal sorting complexes required for transport) through a complex protein network (reviewed in [32]). The regulation of the traffic of endosomal cargo proteins is not only dependent on E3 ligases, but also on two deubiquitinating enzymes with opposite effects, AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) and UBPY, which interact with ESCRT protein components and are relevant for its regulation. AMSH has been proposed to play an editing role, effectively rescuing ubiquitinated substrates from lysosomal degradation by a proof-reading activity. Instead, UBPY, with dual specificity for Lys-63 and Lys-48 chains and with a broad range of targets, by acting on both cargo proteins and ESCRT would protect its substrates from lysosomal and proteasomal degradation [32]. Moreover, UBPY can also form trimeric complexes with another DUB from the OTU (ovarian tumour) gene family (see Table 2) and an E3 ligase, GRAIL [33], then meeting other cellular requirements as yet not fully characterized.

Remarkably, ESCRT complexes, UBPY and AMSH have also been recently involved in cytokinesis [34], thus showing the recruitment of similar complex components for regulating different biological processes. In cytokinesis, ubiquitination rises strikingly but transiently, and at least one of the central spindle SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, VAMP8 (vesicle-associated membrane protein 8), is ubiquitinated by an as yet unidentified E3 ligase to be subsequently deubiquitinated by UBPY and AMSH. These two opposing processes are strictly regulated in both localization and timing in this very dynamic process. Depletion of any of these DUBs by knockdown causes failure to deubiquitinate VAMP8, thus leading to severe cytokinesis defects [34].

The use of alternate antagonistic ubiquitinating and deubiquitinating activities on the same substrate or in different subunits acting on the same complex may be quite a common mechanism in highly dynamic processes. If we focus again on mitosis, the spindle checkpoint prevents chromosome mis-segregation by controlling the APC (anaphase-promoting complex), a Ub ligase that, together with its cofactor Cdc20, promotes sister chromatic separation by inducing degradation of securin and mitotic cyclins (reviewed in [35]). A DUB, USP44 (protectin), prevents premature activation of APC by stabilizing the inhibitory APC complex, Mad2–Cdc20. Once kinetochores are attached to the mitotic spindle, APC multiubiquitination disassembles Cdc20 from the inhibitory complex, resulting in strong APC activation. Therefore a dynamic balance of ubiquitination by APC and deubiquitination by USP44 contributes to the generation of the control of the switch-on/off of anaphase entry [36,37].

Ub and Ubls cross-talk in the regulation of large multisubunit complexes: CRLs (cullin-RING Ub ligases) and PML (promyelocytic leukaemia) nuclear bodies

The CRLs are the largest E3 ligase family (Table 1). CRLs are multisubunit complexes that include a member of the cullins, a RING H2 finger protein, a substrate-recognition subunit, and additionally, an adaptor to link the latter to the complex [38,39]. CLRs are activated by the conjugation of a Ubl (Nedd8) to the cullin subunit, so the corresponding neddylating enzymes and adaptor proteins have to interact, even if transiently. In addition, most CRLs dimerize or even multimerize, by means of homodimerization of one of the complex subunits by conventional protein–protein interacting domains, or by means of UBDs, one UBD in cullin binding the conjugated Nedd8 in another cullin subunit. Dimerization of CRLs is now viewed as a potential regulatory synergistic mechanism for optimal Ub-chain elongation ([39] and references cited therein). The stability and activity of CRLs are also regulated by ubiquitination, as many CRLs undergo autoubiquitination and can be the substrate of other regulatory E3 ligases, thus being targeted to the proteasome.

If Nedd8 is required for the CRL activity, it directly ensues that cullin-deneddylation is an effective means to inhibit CRLs, and this is the mechanism used by the two main inhibitors: the CSN (COP9 signalosome) and CAND1. CAND1 inhibits CRLs by sequestering some subunits and preventing cullin neddylation. CSN is a large multimeric complex associated with multiple activities: phosphorylation, deneddylation and deubiquitination. CSN acts by both inhibiting and stabilizing CRLs: by deneddylation of cullins effectively stabilizes inactive CRLs by preventing their autoubiquitination, but additionally, by deubiquitinating some subunits, stabilizes the complex [38,39].

In brief, bringing together – transiently or not – subunits with opposed activity is a resourceful strategy for the cell to regulate activity and signalling without disposing off the complexes, allowing fast activation/deactivation cycles of CRLs, and responding to a variety of cell requirements.

Most recently, several reports have pointed out that regulation of the PML nuclear bodies depends on a multiple cross-talk between SUMO and Ub. PML can be mono- and poly-SUMOylated. There is a balance between conjugation and deconjugation of SUMO chains in PMLs, depending on the cellular state. When PML is poly-SUMOylated, it is then able to interact with the RFN4 E3 ligase, which by conjugating a poly-Ub chain directs PML to proteasome degradation. Therefore a dynamic balance between mono- and poly-SUMO is required for regulating PMLs, as a shift towards poly-SUMO chains targets PML for polyubiquitination and subsequent proteasome degradation [40,41].

The future

To ensure a finely tuned regulation of the modified substrate, cells have resorted to multifunctional complexes in which one or several Ub (and/or Ubl) ligases and also one or some deubiquitinating (and/or Ubl-deconjugating) enzymes are brought together [42]. In doing so, both the signalling stimuli and the feedback activity within the complex are integrated and exert a tight control on the system by switching-on or -off activities, thus responding promptly to specific cellular requirements. These protein modification complexes should be highly dynamic scaffolds, able to recruit new activities and substrates through different adaptor proteins, and shift subunits at a fast pace in response to cellular stimuli. Besides, and to add further layers of complexity, the same subunit – either E3 ligase, DUB or adaptor – may participate in different complexes, regulating different biological processes, the tempo and the versatility being the operative issues.

What next? In view of the data presented here, a completely new picture emerges for most proteins, particularly those involved in dynamic processes. When looking now at your favourite protein, ask yourself how its turnover, localization and activity regulation might be connected to the Ub and Ubl tags, how it might be recruited to a modifying complex, how and when is your protein going to be recognized for conjugation, and by what, and how and when will it be rescued, shuttled or subtly shifted it from its allotted fate, and by what. Succinctly put, and also for proteins, ‘it all depends on your partners’.

Third Intracellular Proteolysis Meeting: A joint Biochemical Society and INPROTEOLYS Network Focused Meeting held at Auditorio de Tenerife, Santa Cruz de Tenerife, Canary Islands, Spain, 5–7 March 2008. Organized and Edited by Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (Barcelona, Spain), Manuel Rodríguez (CICbioGUNE, Derio, Spain), Eduardo Salido (La Laguna, Tenerife, Spain) and Dimitris Xirodimas (Dundee, U.K.).

Abbreviations

     
  • AMSH

    associated molecule with the SH3 domain of signal transducing adaptor molecule

  •  
  • APC

    anaphase-promoting complex

  •  
  • CSN

    COP9 signalosome

  •  
  • DUB

    deubiquitinating enzyme

  •  
  • ER

    endoplasmic reticulum

  •  
  • ERAD

    ER-associated degradation

  •  
  • ESCRT

    endosomal sorting complexes required for transport

  •  
  • Mdm2

    murine double minute 2

  •  
  • NPC

    nuclear pore complex

  •  
  • OTU

    ovarian tumour

  •  
  • PML

    promyelocytic leukaemia

  •  
  • RING

    really interesting new gene

  •  
  • Ub

    ubiquitin

  •  
  • CRL

    cullin-RING Ub ligase

  •  
  • SUMO

    small Ub-related modifier

  •  
  • UBD

    Ub-binding domain

  •  
  • Ubl

    Ub-like molecule

  •  
  • UBP

    Ub-specific protease

  •  
  • UBPY

    UPB Y

  •  
  • UPS

    Ub/proteasome system

  •  
  • USP

    human Ub-specific protease

  •  
  • VAMP8

    vesicle-associated membrane protein 8

The research work in our group is funded by BFU2007-60823 to G.M., and A.D. is in receipt of a BRD Ph.D. fellowship from the University of Barcelona. We are indebted to Anna Bosch-Comas, Roser Gonzàlez-Duarte and other past and present members of our group for useful discussions.

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