The ubiquitin–proteasome field has matured, as is evident from the wide diversity of systems and mechanisms in which it participates and that are the subject of investigation, presented in the Ubiquitin–Proteasome System, Dynamics and Targeting meeting held in Barcelona, co-sponsored by the Biochemical Society, the Spanish Ministry of Science, the Spanish Research Council and the Catalan Academy of Sciences. Several of the aspects dealt with in the meeting are discussed in detail in the collection of review papers included in this issue of Biochemical Society Transactions. These papers reflect the importance of ubiquitin and ubiquitin-like modifiers as enormously versatile signalling entities that modulate and direct pathways in specific directions through modification-induced interactions. One conclusion from the meeting is that the field has become so rich and dense that, in order to be useful and informative, future meetings may need to focus on particular aspects of the ubiquitin–proteasome system.

Mechanisms of specificity regulating the UPS (ubiquitin–proteasome system)

Acquisition of complexity of the proteasome during evolution can be delineated by a comparative analysis from archaea to eukaryotes. In carrying out this exercise, it is observed that proteasome architecture is conserved, but the number of genes devoted to building the complex has increased, as well as the components of the RP (regulatory particle). A recently discovered evolutionary implementation are the yeast and mammal proteasome chaperones, reviewed by Park et al. [1] (see pp. 6–13). Several studies have shown that Hsm3 (S5b), Nas2 (p27), Nas6 (gankyrin) and Rpn14 [PAAF-1 (proteasomal ATPase-associated factor 1)] are present in intermediate complexes, but not in fully mature proteasomes, and that their mutations cause defects in proteasome association. These chaperones bind the C-terminal tails of RP ATPases and facilitate their insertion into specific pockets in the CP (core particle) α-subunits, thus regulating RP–CP assembly. Moreover, during the process of RP formation, ATPases form pairs before the final ATPase hexameric ring, and proteasomal chaperones are also involved in this process. The RP interacts with polyubiquitylated proteins, specifically tagged for degradation. RP subunits Rpn10 (S5a) and Rpn13 contain UBDs (ubiquitin-binding domains) that bind directly to ubiquitin chains, and subunit Rpn1 interacts with Rad23, Dsk2 and Ddi1, factors that bind polyubiquitylated substrates. Lopitz-Otsoa et al. [2] (pp. 40–45) show the variable distribution of UBDs in protein sequences. UBDs may form tandems in polyubiquitin-binding proteins, such as S5a, RAP80, Rad23 and NUB-1. The higher affinity for polyubiquitin of UBDs in tandem compared with single UBDs prompted the authors to develop artificially fused UBDs [known as TUBEs (tandem disposition of ubiquitin-binding entities)] to generate a powerful tool to purify native polyubiquitylated proteins, reaching affinities for polyubiquitin in the nanomolar range.

Protein modification by SUMO (small ubiquitin-related modifier) is not commonly involved in proteasome-dependent proteolysis. Denuc and Marfany [3] (see pp. 34–39) describe multiple contacts between ubiquitin and SUMO systems and demonstrate a role for SUMO in targeting proteins to the proteasome. STUbLs (SUMO-targeted ubiquitin ligases) recognize SUMO-modified proteins and target them to the proteasome. STUbLs usually contain N-terminal SIMs (SUMO-interaction motifs) and C-terminal RING finger domains. Other examples of ubiquitin–SUMO cross-talk are discussed: E2, E3 and DUBs (deubiquitylating enzymes) are modified and regulated by SUMO [e.g. E2-25K, Mdm2 (murine double minute 2) and USP25 (ubiquitin-specific peptidase 25)], and a novel type of ligase catalyses both ubiquitylation and SUMOylation [TOPORS (topoisomerase I-binding, arginine/serine-rich)]. The proximity of ubiquitin and SUMO signals is also observed in proteins modified by both modifiers in the same lysine residues, but with heterogeneous functional effects {e.g. IκBα [inhibitor of NF-κB (nuclear factor κB) α] and BMAL1 [brain and muscle ARNT (aryl hydrocarbon receptor nuclear translocator)-like 1]}. The NF-κB pathway is controlled by multiple ubiquitin-mediated signals. Recently, it has been proposed that the COP9 signalosome complex, evolutionarily related to the lid of the proteasome, also regulates the NF-κB pathway. Notably, the signalosome, which contains CSN5 (COP9 signalosome subunit 5), a subunit with deubiquitylating activity, would support Lys63-linked ubiquitylation of TRAF2 (tumour-necrosis-factor-receptor-associated factor 2) and RIP (receptor-interacting protein). This interesting interaction and other links between CSN5 and NF-κB regulation are discussed by Schweitzer and Naumann [4] (see pp. 156–161). Deubiquitylation regulates multiple ubiquitin related processes, and DUBs are emerging biomedical targets. In a remarkable effort, Harper and colleagues have developed a technology to find bona fide protein interactors from systematic proteomics analysis and they have applied it to determine the interactome of human DUBs [5]. This work and its implications are discussed by Katz et al. [6] (see pp. 21–28).

SUMO-regulated processes

The conjugation of eukaryotic proteins to SUMOs regulates a wide range of cellular processes. Historically, SUMOylation was associated with nuclear shuttling, formation of PML (promyelocytic leukaemia protein)-containing nuclear bodies and regulation of transcription, but now we know that these functions are just the tip of the iceberg. More recently, a novel role for SUMO in ubiquitin-mediated protein degradation has been uncovered ([3] on pp. 34–39). Ongoing expansion of our technological horizon will lead to novel functional and mechanistic insights into protein SUMOylation.

In plants, approaches used to study SUMOylation include genetic manipulations and biochemical studies. Gene duplication is a common phenomenon linked to the evolution of flowering plants. Members of the Arabidopsis SUMOylation machinery seem to have undergone several duplication events, resulting in a higher level of complexity than in other organisms. As pseudogenes are frequently found in plants and also in many other eukaryotes, it is essential to determine whether all genes are functional. Furthermore, it is important to determine whether different enzymes and SUMO molecules are involved in different processes or act in a redundant manner. Maria Lois [7] (see pp. 60–64) summarizes for us our current knowledge of the mechanistic aspects of the identified machinery components of the SUMOylation system in Arabidopsis.

Until very recently, SUMOylation and ubiquitylation were regarded as different, although sometimes convergent, systems. We now know that both processes are intimately linked, with unpredictable consequences. Talamillo et al. [8] (see pp. 54–59) present a review on the role of SUMO and ubiquitin in the regulation of transcription factors involved in adrenal cortex formation, steroidogenesis and the hormonal response. Both modifications can potentially regulate not only the half-life of transcription factors, but also their localization or directly affect the assembly of transcriptionally active complexes.

Recently, RING finger proteins have been identified in yeast and mammals that ubiquitylate SUMOylated proteins. This appears to be related to the formation of SUMO chains and their binding by SIMs in these RING finger proteins ([3] on pp. 34–39). SUMO chains are also important during replication, mitosis and meiosis. The capacity of SUMO family members to multimerize is discussed in this issue by Alfred Vertegaal [9] (see pp. 46–49).

Ubiquitin and the cell cycle

The UPS represents a major mechanism of cell-cycle regulation which ensures the correct and unidirectional progression of the cell cycle. Degradation of the vast majority of cell-cycle regulators is controlled by two classes of E3 ubiquitin ligase complexes: the SCF (Skp1/cullin/F-box) complex and the APC/C (anaphase-promoting complex/cyclosome). APC/C and SCF act at different moments of the cell cycle. Thus, in a simplistic way, APC is active from halfway through mitosis (anaphase) until the end of the G1-phase, whereas SCF is active between late G1-phase and the beginning of mitosis. In addition to the complexity of the system, SCF and APC/C regulate each other by targeting for degradation some of their components.

In this context, Manchado et al. [10] (see pp. 65–71) review current knowledge about APC/C functions. APC/C is formed, on the one hand, by an invariable core that includes APC11, APC2 and at least ten other subunits with barely defined roles; and on the other hand, by two variable subunits that play an essential role in cellular division: Cdc20 and Cdh1. These subunits interact only transiently with APC/C and confer substrate specificity. Regulation of Cdc20 and Cdh1 is critical for the appropriate APC/C activity. This regulation includes targeted degradation, phosphorylation [APC/CCdh1 is inhibited, whereas APC/CCdc20 is activated by Cdk1 (cyclin-dependent kinase 1) phosphorylation] and interactions with inhibitory proteins. Moreover, the activity of APC/C is negatively regulated by the SAC (spindle assembly checkpoint), a mechanism that detects unattached chromosomes. Once all chromosomes are bipolarly attached, active APC/CCdc20 targets securin and cyclin B for degradation, resulting in inactive Cdk1 and separation of sister chromatids at the onset of anaphase. After Cdk1 inactivation, dephosphorylation of Cdh1 renders an active APC/CCdh1 complex that allows mitotic exit by targeting for degradation Plk1 (Polo-like kinase 1), Aurora kinases and mitotic cyclins. Intriguingly, two bona fide prometaphase APC/C substrates, cyclin A and Nek2A are degraded, while the SAC prevents the degradation of securing and cyclin B by the same complex. van Zon and Wolthuis [11] (see pp. 72–77) discuss how this paradox can be explained. According to the reported models, cyclin A and Nek2A are either extremely efficient Cdc20 substrates or they use ‘stealth’ mechanisms to escape detection by the SAC. The topic of cyclin A degradation is addressed by Mateo et al. [12] (see pp. 83–86) who discusses the role of acetylation. The emerging model suggests that the acetyltransferase PCAF (proliferating-cell nuclear antigen) acetylates cyclin A at Lys54, Lys68, Lys95 and Lys112 during mitosis, thereby facilitating APC/C ubiquitylation and proteasome degradation.

Ubiquitin and DNA-damage repair

The cell response to DNA damage and genotoxic stress is highly regulated, given that many of the pathways activated when the stability or integrity of the DNA is compromised are either mutagenic or trigger cell death by apoptosis. In this context, several mini-reviews have focused on the extremely high complexity that different post-translational modifications to key proteins confer on the cellular response. Translesion synthesis, one of the strategies that the replicating cell uses for DNA-damage tolerance in order to bypass stalled DNA replication forks is one of those highly regulated pathways. Whereas Gallego-Sánchez et al. [13] (see pp. 104–109) reviewed the regulation of mono- and poly-ubiquitylation of yeast PCNA (proliferating-cell nuclear antigen), stressing that not only the ubiquitin ligases, but also the rate and activity of the deubiquitylating enzymes are to be dissected in order to understand how the switch of the polymerases is controlled in the lesion site, Chun and Jin [14] (see pp. 110–115) focus on the ubiquitin-dependent activation and recruitment of the other half of the translesion synthesis, the translesion polymerases themselves. Intriguingly, both reviews envisage a situation in which the simultaneous, but independent, mono-ubiquitylation of the three monomers in the PCNA homotrimer would allow the combinatorial recruitment of distinct polymerase activities with different affinities, to efficiently bypass the lesion with a minimum of errors.

The relevance of post-translational modifications in the control of the DNA-damage response is exemplified by SUMO as well as by covalent ubiquitin modifications. This is why Selvarajah and Moumen [15] (see pp. 87–91) present their proteomic strategy to identify new key DNA-damage proteins. Their approach relies on the differential affinities of UBDs for mono- and poly-ubiquitin modifications, which can be used for the enrichment of endogenous ubiquitylated proteins in lysates from cells under different genotoxic treatments, coupled with gel electrophoresis and protein identification by MS. Also, SUMO modifications are required for the precise regulation of the BRCA1 (breast cancer early-onset 1) DNA-damage response and the DNA replication and repair pathways, particularly for accruing and activating the proteins of the repair complexes to the lesion sites, and, as Morris [16] (see pp. 92–97) points out in her review, the SUMO pathway maintains a fluent dialogue with the ubiquitin pathway, through the SRUbLs (SUMO-regulated ubiquitin ligases) and STUbLs. Thomson and Guerra-Rebollo [17] (see pp. 116–131) present a detailed summary on how a concert between phosphorylation plus ubiquitylation and SUMO modifications exquisitely regulate the recruitment, transient activation, affinity for the lesion and dissociation of essential activities involved in BER (base excision repair), NER (nucleotide excision repair) – in both the transcription-coupled repair and the global genome repair pathways – and the repair of double-strand breaks.

Finally, when focusing on particularly relevant checkpoint proteins in the DNA-damage response, p53 is raised again as a paradigm, but not only because of the multiplicity of post-translational modifications considered on their own, from acetylation and phosphorylation to SUMOylation and ubiquitylation, but rather, as Benkirane et al. [18] (see pp. 98–103) argue in their mini-review, because this multiplicity is the reflection of an all-connecting cellular network, which translates into a cell that can reach easily a new equilibrium once the stress stimuli disappear, in what the authors call a metastable network.

Drug discovery in the UPS

The approval in 2003 by the U.S. FDA (Food and Drug Administration) of the proteasome inhibitor bortezomib (Velcade) for the treatment of multiple myeloma opened an entire new field in drug development, aimed at targeting the UPS. The apparently naïve idea of pharmacologically targeting the UPS [19] crystallized in the development of bortezomib and its use as an anticancer drug [20], in spite of the relatively sparse mechanistic knowledge of the regulation and many functions of the UPS at the time. More than 15 years later, intense research has led to much more detailed information on the structural and biochemical bases of many facets of the cell life cycle and specialized functions regulated by the UPS and ubiquitin-like modifications. This knowledge is being translated by pharmaceutical companies and academic groups into schemes for the discovery and development of drugs that target not only the proteasome, but also specific upstream or downstream components of the system, such as the NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8)-activating enzyme [21], ubiquitin ligases [22,23], deubiquitylating enzymes [24] or ubiquitin turnover [25]. Most of these developments are intended for cancer therapy, although other pathologies, including neurodegenerative disorders and infectious diseases, are increasingly being recognized as potential beneficiaries of treatment by drugs that modulate the UPS at different levels.

In their paper, Celia Berkers and Huib Ovaa [26] (see pp. 14–20) discuss the need for less toxic proteasome inhibitors directed at specific enzymatic activities of this multisubunit protein assembly, and focus their review on the current approaches for the design of activity-based assays to search for bioavailable and specific inhibitors of the proteasome. They also discuss the mechanisms of activity and the cellular effects of recently developed compounds that target specific subunits and catalytic centres of the proteasome and the immunoproteasome.

Goldenberg et al. [27] (see pp. 132–136) present an update on activity-based assays designed to discover ubiquitin ligase inhibitors, including reactions in which the components are either unbound or immobilized, and the ubiquitylation reaction is monitored by FRET (fluorescence resonance energy transfer), ELISA or with radioactive probes. As discussed by them, drug developers currently favour screening for inhibitors of the HECT (homologous with E6-associated protein C-terminus) domain ligases that have relatively more druggable catalytic activities than RING finger domain ligases that function through tethering E2s to substrates through protein–protein interactions, in part because of the widespread conviction that protein–protein interactions are far more difficult to target in a cost-effective manner in HTS (high-throughput screening) schemes. In spite of this, the most successful ubiquitin ligase inhibitor to date is precisely a protein–protein interaction antagonist that inhibits the Mdm2-directed degradation of p53 [20].

Frédéric Colland [28] (see pp. 137–143) discusses deubiquitylating enzymes as drug targets. The rationale here is to confer enhanced sensitivity to proteasome-dependent degradation on specific substrates by inhibiting substrate-specific DUBs, quite different from proteasome inhibitors that seek to enhance protein stability, for example the NF-κB inhibitor IκB in the case of bortezomib. Colland emphasizes the work of his own group to develop inhibitors for USP7, a DUB that stabilizes Mdm2 [a ligase for the tumour suppressors p53 and FOXO4 (forkhead box O4)] or SCF–βTrCP (β-transducin repeat-containing protein; a ligase for β-catenin, claspin and many more signalling and cell-cycle regulators). Specific drugs can be used for prevention or treatment of viral infection. In this sense, the review by Campagna and Rivas [29] (see pp. 50–53) on the reported antiviral action of the well-known anti-aging compound resveratrol, most probably through p53, NF-κB or PML molecular pathways, is suggestive of another potential pharmaceutical target.

All of these review papers highlight how rational approaches can be devised to exploit the enormous potential offered by specific USP components as targets for the discovery and development of novel classes of drugs to fight cancer, neurodegenerative disorders, inflammation or viral infections.

UPS, autophagy and neurodegenerative disorders

The two major systems for the intracellular disposal of proteins are the UPS and autophagy [29]. Although the regulated degradation of proteins mainly proceeds through the UPS, bulk degradation of proteins that are misfolded and are resistant to UPS is handled through the autophagy–lysosome pathway, as well as the ERAD (endoplasmic-reticulum-associated degradation) pathways, closely associated with the UPS [30]. Excessive accumulation of misfolded proteins caused by mutations in the proteins, or the impairment in any of the systems dedicated to the turnover of misfolded proteins, trigger the ER stress response [UPR (unfolded protein response)] in an attempt to cope with these misfolded proteins. Failure to degrade these proteins results in the formation of pathological aggregates and eventual cell death, all of these processes underlying the aetiology of most neurodegenerative disorders. It is therefore of the utmost interest to understand the mechanisms that regulate these protein degradation pathways in order to design better therapies for these highly disabling and intractable diseases.

Ground-breaking work from the Ohsumi laboratory [31,32] has established that autophagy and the biogenesis of autophagosomes is directed by the autophagy pathway, which bears striking similarities to the biochemical pathways that direct the modification of proteins by ubiquitin. Until recently, the evidence for a relationship between the UPS and autophagy was limited to the observation that autophagy would handle excess unfolded proteins that could not be processed by the UPS. Recent work has unveiled the existence of much tighter mechanistic links between the UPS and autophagy that coordinate these two seemingly separate protein degradation systems. It has been shown that autophagy functions as a compensatory system for the proteasome when the latter is impaired, and that the undegraded polyubiquitylated proteasome substrates are targeted to autophagosomes by HDAC6 (histone deacetylase 6), a ubiquitin- and microtubule-binding deacetylase [33]. The Rubinsztein laboratory has found that inhibition of autophagy compromises the proteasomal degradation of proteins as a consequence of an accumulation of p62 (sequestosome, or A170/SQSTM1) [34]. p62, together with NBR1, bind Lys63-chain polyubiquitylated proteins and target them to autophagosomes [35].

Chin and colleagues [36] (see pp. 144–149) review the role played by the aggresome–autophagy pathway in the clearance of misfolded proteins and how neurodegeneration can be caused either by dysfunction of these systems or by chronic overload with misfolded proteins, for example caused by mutations in certain proteins. As stated above, polyubiquitylation by Lys63-based chains is critical for the targeting and subsequent processing of misfolded proteins by autophagosomes, and Chin et al. [36] review the evidence furnished by them and others that the RING finger protein Parkin is a critical ubiquitin ligase for this modification, in conjunction with the UBC13 (ubiquitin-conjugating enzyme 13)–UEV1 (ubiquitin-conjugating enzyme variant 1) heterodimeric ubiquitin conjugating enzyme, and discuss further the mechanistic, diagnostic and therapeutic relevance of the accumulation of Lys63-linked polyubiquitin chains in the aggregates associated with neurodegenerative disorders, particularly Parkinson's disease. Haapasalo et al. [37] (see pp. 150–155) discuss the role played by the UBA (ubiquitin-associated)- and UBL (ubiquitin-like)-containing protein ubiquilin-1 in the formation of the amyloid plaques associated with AD (Alzheimer's disease) and in the turnover of wild-type and mutant forms of presenilin. Because ubiquilin-1 associates with aggresomes in AD samples, it is speculated that it might also participate in their targeting to autophagosomes.

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)

Abbreviations

     
  • AD

    Alzheimer's disease

  •  
  • APC/C

    anaphase-promoting complex/cyclosome

  •  
  • Cdk1

    cyclin-dependent kinase 1

  •  
  • CP

    core particle

  •  
  • CSN5

    COP9 signalosome subunit 5

  •  
  • DUB

    deubiquitylating enzyme

  •  
  • IκB

    inhibitor of nuclear factor κB

  •  
  • Mdm2

    murine double minute 2

  •  
  • NF-κB

    nuclear factor κB

  •  
  • PCNA

    proliferating-cell nuclear antigen

  •  
  • PML

    promyelocytic leukaemia protein

  •  
  • RP

    regulatory particle

  •  
  • SAC

    spindle assembly checkpoint

  •  
  • SCF

    Skp1/cullin/F-box

  •  
  • SUMO

    small ubiquitin-related modifier

  •  
  • SIM

    SUMO-interaction motif

  •  
  • STUbL

    SUMO-targeted ubiquitin ligase

  •  
  • UBD

    ubiquitin-binding domain

  •  
  • UPS

    ubiquitin–proteasome system

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