The International Journal of Biochemistry & Cell Biology 40 (2008) 1126–1140
Available online at www.sciencedirect.com
Review
Alternative UPS drug targets upstream the 26S proteasome
Roland Hjerpe, Manuel S. Rodrıguez ∗Ubiquitin-Like Molecules and Cancer Laboratory, Proteomics Unit, CIC bioGUNE, CIBERehd,
Bizkaia Technology Park, Building 801A, 48160 Derio, Spain
Available online 8 December 2007
Abstract
The successful use of proteasome inhibitors in anti-cancer therapy encouraged the development of new drugs targeting theactivity of the ubiquitin-proteasome system (UPS) at various levels. The UPS comprises a complex set of protein adaptors whosecoordinated function modulates the interaction of ubiquitin-modified proteins with protein effectors. In addition, UPS crosstalkwith other post-translational modifications, complicates a sophisticated set of conjugation and de-conjugation pathways, providinga large variety of potential targets for the development of specific inhibitors. Traditionally associated with the proteasome, ubiquitin-conjugation does not always result in protein degradation. The major signal that targets proteins for degradation is the formationof ubiquitin-chains on lysine 48 (K48). Other ubiquitin features such as K63 chains or mono-ubiquitylation appear to regulate thetransient formation of functional macromolecular complexes or relocate modified proteins inside the cell. The emerging idea thatUPS-mediated degradation participates not only in the initiation but also in the termination step of certain functions, highlights new
Multiple post-translational modifications act to regu-ate the activity of many essential cellular factors. These
odifications facilitate a rapid cellular response, alter-ng the activity of the target proteins by modifyingheir capacity to associate with multiple partners, theirnteraction with downstream factors, their sub-cellularocalisation or by promoting variations in protein syn-hesis and stability. Amongst the different strategies toontrol protein activity, probably the most drastic ones protein degradation, because it inactivates all proteinunctions. In eukaryotic cells, the ubiquitin-proteasomeystem (UPS) drives one of the most important prote-lytic activities (Fang & Weissman, 2004). In the UPS,roteolysis mediated by the 26S proteasome is condi-ioned by the previous modification of the substrate withbiquitin. As its name implies, ubiquitin is a ubiquitousnd highly conserved protein found in all eukaryoticissues. The ubiquitin molecule can be found free oronjugated to protein substrates, where it drasticallyodifies the biochemical properties of the target pro-
ein. The active form of ubiquitin is generated from aigh molecular weight precursor by the action of ubiq-itin C-terminal hydrolases (UCH), which release theature 8 kDa protein. After cleavage, ubiquitin exposes
lycine 76, which is involved in the isopeptide bond for-ation with a lysine residue on target substrates. When
onjugated, ubiquitin exists as a monomer or as poly-ers, forming chains of ubiquitin, using seven internal
ysine residues (K6, K11, K27, K29, K33, K48 and63). Depending on the number of ubiquitin molecules
ttached to the protein substrate and the lysine residuesn the ubiquitin moieties involved in the formation ofbiquitin chains, the destiny of a protein will be dif-erent. The in vivo significance of ubiquitin chains haseen the most studied in the cases of K48 and K63 poly-biquitin (Fang & Weissman, 2004). When conjugationccurs through lysine 48, proteins appear to be targetedainly to proteasome-mediated degradation. It has been
roposed that at least four molecules of ubiquitin are
equired for degradation by the proteasome (Thrower,offman, Rechsteiner, & Pickart, 2000). In contrast,ono-ubiquitylation or conjugation trough lysine 63 are
nvolved in processes as diverse as endocytosis, trans-
lation, DNA repair or activation of signal transductionpathways (Mukhopadhyay & Riezman, 2007; Murata &Shimotohno, 2006; Sun & Chen, 2004).
With the large number of target proteins and cellularfunctions regulated by the ubiquitin pathway, a posi-tive outcome from the use of the proteasome inhibitorbortezomib (Fig. 1) in cancer therapies was somewhatsurprising. The rationale of this achievement is proba-bly based on the hyperactivity of cancer cells, comparedto non-tumoural ones, in which the deregulated activ-ity of critical checkpoint factors results in the loss ofcell division control typically found in tumour cells (fora recent review see Armand et al., 2007). Recent datasuggest that part of the cytotoxic effects of proteasomeinhibitors could be related to their ability to inhibit pro-tein synthesis, as different proteasome inhibitors wereobserved to lead to a dose-dependent inhibition of pro-tein synthesis, followed by cell death. Although the effectof proteasome inhibitors on protein biogenesis is clear,the exact molecular mechanisms leading to these effectsrequire further efforts to be clarified (Ding, Dimayuga,Markesbery, & Keller, 2006; Stavreva et al., 2006). Aninteresting hypothesis is that defective ribosomal prod-ucts (DRiPs) may be responsible for cytotoxic effectsobserved after proteasomal inhibition (Ding, Cecarini,& Keller, 2007). During the translational process, thereis a continuous production of DRiPs (Yewdell, Anton,& Bennink, 1996), due to physiological protein misfold-ing and premature translational termination. DRiPs are amajor substrate for the ubiquitin-proteasome system, andit has been suggested that these poly-peptides may pro-vide a link between protein synthesis and degradation.An overall lower rate of protein synthesis during pro-teasomal inhibition might in addition lead to a decreaseof factors essential for cell viability, proteasomal sub-strates and components of the UPS. The fact that thereis a crosstalk between the proteasome and the ribosome,connecting protein synthesis with protein degradation,adds another aspect to consider when inhibiting the pro-teasome. Although, perhaps it is not surprising that thereis a connection between the UPS and the ribosome, con-
sidering the fact that a main bulk of mature ubiquitinin yeast derives from ribosomal ubiquitin fusion pro-teins (Chan, Suzuki, & Wool, 1995; Finley, Bartel, &Varshavsky, 1989).
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Fig. 1. Different levels for blocking the UPS. Each of the enzymes involved in the conjugation, de-conjugation, or proteins connecting with effectorfunctions, including the presentation of substrates to the proteasome can be considered as a potential point for intervention (indicated with a stop sign).(A) The E1 inhibitors Panepophenantrin and Himeic Acid (Sekizawa et al., 2002; Tsukamoto et al., 2005). (B) Small molecule E2 inhibitors, whichcompete with UEV1 for interaction with UBC13 (Thomson, personal communication). (C) Hypothetical point of intervention with the assemblyof SCF ubiquitin ligase complexes. (D) Inhibitors of Mdm2-p53 interaction exemplifying interruption of E3-substrate interaction—examples areNutlins (Vassilev et al., 2004), RITA (Issaeva et al., 2004), benzodiazepinediones (Koblish et al., 2006) and Chlorofusin (Lee et al., 2007). (E)Hypothetical point of intervention with E4 enzymes. (F) DUB peptide inhibitors, preventing the de-ubiquitylation of a substrate (Borodovsky et al.,2005). (G) Ubistatins, which inhibit interaction of ubiquitin receptor proteins with the hydrophobic surface patch of ubiquitin (Verma, Peters, et al.,2004). (H) examples of proteasome inhibitors—the peptide boronate bortezomib, which is in clinical use, the peptide aldehyde MG132, lactacystinand the peptide epoxyketon epoxomycin (Borissenko & Groll, 2007).
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The most intriguing question which needs to benswered in order to understand the positive effectsf proteasome inhibition observed in clinical trials, ishy these effects are not observed for all types of
ancer? The simplest answer to this question is thateregulated factors most certainly are not the same.herefore, pharmacodynamics leading to positive effectsfter proteasome inhibitor treatment might be differentepending on the molecules at the origin of a particularathological process. With this logic it becomes neces-ary to better determine the origin of the pathology at aolecular level, to be able to identify more appropriate
nd specific treatments. After presenting a short surveyf some potential intervention levels upstream of the pro-easome, we will focus on possibilities of intervention inhe context of cellular functions, such as signalling andranscription, using as main examples the transcriptionactors NF-�B and p53.
. Possible levels of intervention in the UPS
Various possibilities of intervention can be consid-red throughout the UPS. The most obvious targetspstream the proteasome are at the level of ubiquitinonjugation and de-conjugation (Fig. 1). A number ofxcellent reviews describe most possibilities of target-ng ubiquitin conjugating and de-conjugating enzymesDikic, Crosetto, Calatroni, & Bernasconi, 2006; Nalepa,olfe, & Harper, 2006; Nicholson, Marblestone, Butt,
Mattern, 2007) and for this reason many of theselternatives will not be deeply described in this docu-ent. The attachment of ubiquitin to a protein-substrate
s achieved by a cascade of thiol–ester reactions, medi-ted by a ubiquitin activating enzyme (E1), a ubiquitinonjugating enzyme (E2) and a ubiquitin ligase (E3)Hershko, Heller, Elias, & Ciechanover, 1993). For somebiquitin-conjugation reactions, it has been proposedhat the action of an E4 enzyme allows the extensionf ubiquitin chains (Hatakeyama, Yada, Matsumoto,shida, & Nakayama, 2001; Koegl et al., 1999). The mostpstream UPS target for intervention is therefore the1. Two examples of E1 inhibitors are Himeic acid andanepophenantrin (Fig. 1), which block the formationf the E1-ubiquitin intermediate in the first step of thehiol–ester cascade (Matsuzawa et al., 2006; Tsukamotot al., 2005). However, the effect of inactivation of the1 predicts pleiotropic effects even under conditions ofartial inhibition. In fact, temperature sensitive muta-
ions of the ubiquitin E1 leads to cell cycle arrest in theate S phase and G2, underlining the role of ubiquity-ation in cell division and the difficulty to act at thisevel (Finley, Ciechanover, & Varshavsky, 1984; Kulka
et al., 1988). Continuing with the cascade, E2s appearto be more appropriate targets than the E1, as the num-ber of processes affected by the blockade of a singleE2 will be reduced. The first documented interventionat this level concerns Ubc13, which will be discussedin Section 4. Ubiquitin ligases have been considered asprivileged targets for drug design due to their capacityto specifically recognise protein substrates and to trans-fer ubiquitin molecules to a lysine-residue on the target(Nakayama & Nakayama, 2006; Nalepa et al., 2006).Designed drugs blocking E3s most take in considera-tion structure and biochemical activity of these enzymes,which can be classified in two categories: (a) E3s formingthiol–ester intermediates with ubiquitin which includesthe family of HECT E3s. These ligases, characterisedby the presence of the homologous to the E6AP car-boxyl terminus (HECT) domain, were first described inthe complex made up of the Human Papillomavirus 16(HPV16) E6 protein and the cellular E6-associated pro-tein (E6AP), which together target p53 to degradationduring HPV16 viral infection. In this class of enzymes,the C-terminus harbours the active cysteine, which par-ticipates in the transfer of ubiquitin to the target protein,while the N-terminus is responsible for the recognition ofthe substrates. Thus, pharmacological inhibitors of thiscategory of enzymes should take into account these twocharacteristics. (b) E3s that bridge E2s to protein sub-strates, without forming intermediates with ubiquitin. Inthis class of E3s we find the family of U-box and RINGfinger ligases, which are formed of a single or multipleinter-regulated sub-units. Multiple subunit RING fingerE3s represent the largest class of ubiquitin ligases, withmore than 300 human genes encoding RING-finger likeproteins (Petroski MD 2005). E3s of the Skp1-Culling-Fbox (SCF) type consists of a scaffold like cullin, whichintegrates a RING finger containing module (RBX),an adaptor protein (SKP1) and an F box containingprotein, which recognize the substrate (Nakayama &Nakayama, 2006). Five cullin-based ubiquitin E3s havebeen reported and named SCF1, SCF2, SCF3, SCF4 andSCF5 according to the number attributed to each cullin.Their activity depends on the participation of differentE2s and other constituents of the complex. However,a main difference between different SCF ligases is theF-box subunit, which discriminates between protein tar-gets. Although there has been tremendous effort doneto understand specificity, the number of F-box mod-ules which unambiguously recognise a single protein
target, remain limited. More importantly, it is not wellunderstood how F-box proteins having several differ-ent targets can discriminate between them, as is the casefor �-transducin repeat-containing protein (�TRCP) (see
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Section 4). Obtaining this information will be essen-tial to envisage the development of specific inhibitors.A superior level of complexity, but also putative pointfor intervention, concerns the regulation of the activityof SFC and other RING finger ligases by conjugation/de-conjugation of ubiquitin-like modifiers, such as Nedd8and SUMO. Neddylation conditions the association ofcullins proteins with RBX (Nalepa et al., 2006). In thisprocess, CAND1 (cullin-associated and neddylation-associated protein) plays a regulatory role by interferingwith the neddylation of cullins and therefore their asso-ciation with the rest of the subunits required to generatea functional SCF E3. While neddylation involves ubc12and APP-BP1/UBA3 enzymes, deneddylation is medi-ated by the CSN5 subunit of the COP9-signalosomecomplex. The assembly/disassembly of cullin-based E3sappear to condition the in vivo activity of these enzymes.Neddylation also regulates single subunit RING fingerligases such as the p53 ligase Mdm2, which is also sub-strate for SUMOylation and ubiquitylation (Xirodimas,Chisholm, Desterro, Lane, & Hay, 2002; Xirodimas,Saville, Bourdon, Hay, & Lane, 2004). Although someinformation is available, more efforts are required todecipher this intriguing interconnected network of pro-tein modifiers. Other categories of ligases, such as theU-box ligases, whose RING-like structure does not relyon a metal ion, as is the case for RING finger proteins, orthe APC/C (anaphase-promoting complex/cyclosome)type, are also attractive intervention objectives. APC/CE3s are composed by APC11, a RING finger subunit,the APC2 scaffolding protein and protein receptors suchas CDC20 and CDH1. Therefore, they are structurallyrelated to SCF complexes, but include more than 10 com-ponents without a clear role. Interfering with the bindingof the E3 to the substrate, association of the E3 with theE2 or the formation of the thiol–ester intermediates withubiquitin, can be envisaged as strategies to tackle theseenzymes (Fig. 1).
E3s inhibitors are already a reality for the p53 ubiq-uitin ligase Mdm2. Cis-imidazoline molecules calledNutlins have been identified from chemical libraries asmolecules able to block the p53/Mdm2 interaction byoccupying the p53 binding pocket of this E3 (Vassilevet al., 2004). Consequently, Nutlins are able to acti-vate p53-dependent cell cycle arrest, induce apoptosis incancer cell lines, and reduce tumour xenograft growth.However, it should be kept in mind that a recent reportshowed that Nutlins induce G1 cell cycle arrest in a
p53 deficient cell line (VanderBorght et al., 2006). Thisindicates that these molecules may compete with pro-teins other than p53 for Mdm2 binding. An alternativeapproach to the development of E3 inhibitors is to iden-
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tify molecules that bind the protein substrate instead, asthis will avoid effects on various potential “clients” of“promiscuous” enzymes. For example, RITA (2,5-bis(5-hydroxymethyl-2-thienyl)furan) binds the N-terminus ofp53 and promotes growth arrest (Rubinstein et al., 1990;Issaeva et al., 2004). However, the action of RITA alsointerferes with the interaction of p53 with regulatoryfactors such as the p53 cytoplasmic retention proteinPARC (see Section 5) and the acetylating/ubiquitin E4factor protein p300. Other inhibitors such as benzodi-azepinediones (Koblish et al., 2006) and chlorofusin(Lee, Clark, & Boger, 2007) also modulate the activity ofMdm2 towards p53. A detailed description of these andother ways to inhibit Mdm2 has recently been reviewed(Vassilev, 2007).
After substrate degradation occurs, or when aubiquitin-mediated function ends, ubiquitin moleculesare recycled by de-ubiquitylating enzymes (DUBs)that remove ubiquitin moieties from modified pro-teins (Johnston & Burrows, 2006). The human genomeencodes around 95 putative DUBs of which 58 are ubiq-uitin specific proteases (USP), 4 ubiquitin C-terminalhydrolases (UCH), 5 Machado Joseph disease pro-teases (MJD), 14 otubain proteases (OTU) and 14JAMM (JAB1/MPN/Mov34 metalloenzyme) (Nijmanet al., 2005). Except for JAMM proteases, all DUBsare cystein proteases and depend on the catalytic triadformed by cysteine, histidine and aspartic acid residuesto perform the reaction. Inhibitors of enzymes pro-moting the reverse reaction of ubiquitylation wouldresult in a decreased stability and/or activity of themodified protein. Some DUBs have been consideredas potential therapeutic targets because they behaveas oncoproteins or tumour suppressors (Nicholson etal., 2007). USP7 (HAUSP) regulates the ubiquitylationof the RING finger Ubiquitin-E3 Mdm2, which tar-gets p53 to proteasome-mediated degradation. AlthoughUSP7 also de-ubiquitylates p53, it preferentially de-ubiquitylates Mdm2 because of its higher affinity forthe latter substrate (Hu et al., 2006). Mdm2 is addi-tionally de-ubiquitylated by USP2a, which, in contrastto USP7, does not affect the ubiquitylation levels ofp53 (Stevenson et al., 2007). Inhibition of USP7 and/orUSP2a is therefore an interesting strategy to reducethe available levels of Mdm2, which will result in anincreased tumour suppressor activity in cells expressingWT p53.
The fact that a particular substrate may be modified
by mono-ubiquitin or different chain types of poly-ubiquitin calls for the existence of what may be referredto as “ubiquitin receptors”. Such receptor proteins woulddistinguish between the different types of ubiquitin mod-
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fications, and mediate a function depending on thearticular modification and/or modified protein. Theseunctions may or may not be directly related to pro-easomal degradation, but will certainly take part in
odulation of the activity of ubiquitylated proteins byegulating their connection with effector functions. Theroperty of ubiquitin binding is most often localized tomodular domain, which independently can recognize
nd interact with ubiquitin. The first protein motif char-cterized to bind ubiquitin non-covalently was found inhe proteasomal subunit S5a (rpn10 in yeast) (Young,everaux, Beal, Pickart, & Rechsteiner, 1998). S5a is aart of the 19S proteasome regulatory particle, where itinks the base to the lid. The ubiquitin binding propertyf S5a pertains to two independent Ubiquitin Interactingotifs (UIMs), which are short � helical regions. Apart
rom the UIM’s, S5a contains a VWA (von Willebrand A)omain in the N-terminus, which is thought to mediate5a-proteasome interaction (Kang, Chen, Lary, Cole, &alters, 2007). After the discovery of the UIMs in S5a,
ioinformatics approaches identified these motifs in aide variety of proteins with various cellular functions.ollowing the discovery of the UIM, many other ubiq-itin binding domains (UBDs) were characterized, andoday at least 16 different motifs have been described.hese are the UBA, MIU, DIUM, CUE, NZF, A20 ZnF,BP ZnF, UBZ, UEV, PFU, GLUE, GAT, Jab/MPN,BM and the Ubc (Hurley, Lee, & Prag, 2006). Amongst
hese motifs, the UIM and the UBA domain (Ubiquitinssociated domain) are the two best characterized. An
mportant question concerning the trafficking and pre-entation of modified proteins to the proteasome is theay these processes are regulated. Understanding howodified substrates are brought to the proteasome for
ubsequent degradation may result in the identificationf new points of possible therapeutic intervention.
. Interfering with delivery of substrates to theroteasome
UBA domains are classified into four different groups,epending on their ubiquitin binding properties. Class 1nd 2 are defined as binding K48 and K63 poly-ubiquitinhains, respectively, class 3 does not bind poly-ubiquitinhains, and class 4 does not exhibit any particular speci-city for chain linkage in poly-ubiquitin, whilst bindingqually strongly to monoubiquitin (Raasi, Varadan,ushman, & Pickart, 2005). The UBA domain and the
IM are found in many different proteins with diverse
unctions. UBA domains have poor sequence conser-ation, but are well preserved structurally as compacthree helix bundles. In some UBA containing proteins,
a ubiquitin-like domain (UBL) can also be found. Thesedomains share homology with ubiquitin, and many havebeen shown to interact with the proteasome throughS5a (Mueller & Feigon, 2003; Riley, Xu, Zoghbi, &Orr, 2004; Walters, Kleijnen, Goh, Wagner, & Howley,2002). The interaction between S5a and ubiquitin isdependent on a hydrophobic surface patch on ubiquitin.Indeed, mutagenesis of two amino acids (V8A, I44A)in this patch abolished ubiquitin binding by S5a, andalso inhibited proteasomal degradation (Beal, Deveraux,Xia, Rechsteiner, & Pickart, 1996). In addition to S5a,all other helical UBDs have also been reported to inter-act with ubiquitin through its hydrophobic surface patch.hHR23A and hHR23B (human homologs of yeast pro-teins Rad23A/B) are examples of UBA/UBL proteins,containing an N-terminal UBL domain and two UBAdomains. The function of hHR23A may be regulated bycompetition/cooperation arising from intramolecular orintermolecular interactions between its UBAs and UBL.Intramolecular UBL-UBA binding has been suggestedto result in a closed domain organization, which maybe opened up by binding of S5a UIMs to the hHR23AUBL, disrupting hHR23A intramolecular contacts (Mor-ris & Divita, personal communication; Walters, Lech,Goh, Wang, & Howley, 2003). The yeast counterpart ofhHR23A, Rad23, has been shown to be necessary forthe proteasomal degradation of certain substrates whenthe UBL/UBA protein Dsk2 (hPLIC1/2) also is miss-ing (Rao & Sastry, 2002). This suggests a functionaloverlap between UBA/UBL proteins in proteasomaldelivery. Indeed, mutating either rad23 or rpn10 (humanS5a) leads to moderate effects, while a double mutationexhibit strong phenotypes and accumulation of multi-ubiquitylated proteins (Lambertson, Chen, & Madura,1999; Madura, 2004). Moreover, poly-ubiquitylated Tau,a microtubule associated protein involved in neurode-generative disease, requires the UBA/UBL protein p62for being shuttled to the proteasome (Babu, Geetha,& Wooten, 2005). Further, the CDC48UFD1/NPL4/UFD2complex, implicated in ER associated degradation, hasbeen suggested to cooperate with Rad23/Dsk2 in pro-teasomal targeting of substrates (Richly et al., 2005).Among others, all these observations support the notionof UBA/UBL mediated trafficking of specific sub-strates to the proteasome. hHR23A has been reportedto affect the stability of the tumour suppressor p53in various ways. Overexpression of hHR23A stabilizesp53, while hHR23A depletion may lead to a more
rapid p53 degradation or to protection from degrada-tion (Brignone, Bradley, Kisselev, & Grossman, 2004;Glockzin, Ogi, Hengstermann, Scheffner, & Blattner,2003). These results are seemingly inconsistent with a
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role for hHR23A in shuttling ubiquitylated p53 to theproteasome. However, hHR23A and Mdm2 have beenshown to interact, and it has been proposed that protea-somal hHR23A binds p53 when in complex with Mdm2,thus guiding p53 into the proteasome (Brignone et al.,2004). Recently, Kaur et al. show a role for hHR23B inmediating specific interactions of ubiquitylated p53 withchromatin, possibly leading to the initiation of the tran-scription mediated apoptotic response (Kaur, Pop, Shi,Brignone, & Grossman, 2007).
Due to their involvement in the regulation of protea-somal degradation of different factors, the UBL/UBDproteins and their interaction with modified substrates,ubiquitin ligases and proteasomal subunits may consti-tute a plausible target for specific intervention in futuretherapies. Indeed, a first example of small moleculeswhich interfere with the UBD-ubiquitin interaction arethe ubistatins (Verma, Peters, et al., 2004) which targetthe hydrophobic surface patch in the ubiquitin–ubiquitininterface of K48 linked poly-ubiquitin. It was shownby Verma et al. that the ubistatins abolish binding ofSaccharomyces cerevisiae ubiquitin receptor proteinsRpn10 and Rad23 to UbSic1 (recombinant Sic1, ubiq-uitylated in vitro by SCFCDC4) (Verma, Oania, et al.,2004), thus inhibiting its proteasomal degradation. How-ever, the ubistatins are precluded from clinical use dueto their negative charge, which impedes penetration ofthe cell membrane.
Many different ubiquitin E3’s have been reportedto associate with subunits of the proteasome, openingup the possible mechanism of E3 mediated delivery ofsubstrates to this proteolytical machine. Ligases mayconstitute an integral part of the proteasome, or betransiently associated—exemplified by the HECT lig-ase Hul5 (Crosas et al., 2006; Leggett et al., 2002; You& Pickart, 2001) and Mdm2 (Sdek et al., 2005), respec-tively. Mdm2 has been observed to mediate degradationof the retinoblastoma protein (Rb) by the 20S protea-some, in an ubiquitin-independent manner (Sdek et al.,2005). Rb plays an essential role in tumour suppressionin the G1 checkpoint, where it may block entry intoS-phase and thus cell proliferation. Inactivation of Rbfunction is observed in many human malignancies, eitherby Rb mutation or by deregulation of upstream effectors(Giacinti & Giordano, 2006). The ubiquitin independentdegradation of Rb is reliant on a natively folded Mdm2RING domain, as the ubiquitylation-inactive C464AMdm2 mutant no longer can promote Rb degradation,
or the necessary interaction with the proteasomal sub-unit C8 (�7). However, rather than promoting ubiquitinmodification, it appears that the RING motif conforma-tion is important for the interaction with the C8 subunit.
iochemistry & Cell Biology 40 (2008) 1126–1140
The model proposed suggests a bridging mechanism forMdm2, stringing Rb to the proteasome, and thus induc-ing its degradation. Further, the fact that Rb was seen tointeract in vivo exclusively with the 20S subset of pro-teasomes underlines the fact that critical cellular factorscan be targeted for degradation by alternative routes, andthat the Mdm2-C8-Rb interaction might serve as a spe-cific pharmaceutical target for the treatment of humanneoplasms.
Further examples of ligases reported to bind to pro-teasomes include VHL (Corn, McDonald, Herman, &El-Deiry, 2003), APC (Seeger et al., 2003), Parkin(Sakata et al., 2003), SNEV (Loscher et al., 2005),SCFCDC4 (Peng et al., 2003) and TRAF6 (Bouwmeesteret al., 2004)—suggesting that E3 interaction with theproteasome may be a general way for ligases to shuttletheir substrates for degradation. Whether this degrada-tion is mediated by ubiquitin or not may be dependenton each particular ligase and its substrate. Disruptingthe interaction between proteasomal subunits and ubiq-uitin E3’s might constitute a novel way to modulate thedegradation of specific factors.
4. Regulation of signal transduction pathways
The activation of many signalling cascades is regu-lated by ubiquitin and ubiquitin-like molecules and isoften intimately linked with the action of the protea-some. Several signal transduction pathways appear tobe regulated by ubiquitylation. These include TGF�,JAK/STATs, MAPK, and NF-�B pathways. The NF-�Bpathway is so far one of the best characterised ubiquitin-dependent signalling cascades and shows an intriguingcomplexity. As a main regulator process of the NF-�Bactivation, ubiquitylation acts at different levels of thissignalling pathway. Stimulation of cells by TNF�, IL-1�or TCR receptors activates various ubiquitin conjugat-ing and de-conjugating enzymes (Fig. 2). While the E3ligases TRAF2, TRAF5, TRAF6, MALT1 and �-TrCPcan activate this pathway, the ubiquitin-ligase activityof A20, c-IAP1, Itch/Nedd4 and SOCS1 can repressit (reviewed in Krappmann & Scheidereit, 2005). Thereverse reaction driven by CYLD, A20, TRABID andCezanne de-ubiquitylating enzymes contribute to mod-ulate the activation status of this signalling cascade. Theactions of many of these enzymes converge to or emergefrom a main roundabout: the IKK complex, which dic-tates the ubiquitin-mediated proteolysis of the natural
I�B inhibitors of NF-�B. For this reason each of thepreviously mentioned enzymes can be considered aspotential targets for intervention. One of the first Ubiqui-tin E3s to be considered as such was �-TrCP, because its
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Fig. 2. Possibilities for intervention in the ubiquitin-mediated activation of the NF-�B pathway. The ubiquitin-mediated activation of the NF-�Bs rs invoM ugationu favourp
cDKdRScatescs(cemtuT
ignal transduction pathway by the TNFR1, IL1R and TCR receptoALT1, �-TrCP, and A20. Activation of TAK1 and IKK requires conj
biquitin K63 chain de-conjugating activity of A20 acts on RIP1 andoints for intervention.
apacity to recognise the IKK phosphorylated consensusSG(X)2+nS on I�B� and ubiquitylate this inhibitor on21, K22 promoting its proteasomal-mediated degra-ation (Baldi, Brown, Franzoso, & Siebenlist, 1996;odriguez et al., 1996). Degradation mediated by theCF-�-TrCP ligase is mediated by K48 poly-ubiquitinhains as is the one achieved by A20, c-IAP1, Itch/Nedd4nd SOCS1 on RIP1, TRAF2, BCL10 and p65, respec-ively (Scheidereit, 2006). The fact that the �-TrCPnzyme is also involved in the degradation of moleculesuch as EMI1/2, WEE1A, CDC25A/B and �-Catenin,alls for additional studies to better understand the con-equences of the inhibition of such F box proteinsreviewed by Nakayama & Nakayama, 2006). If anyofactor is specifically required for the recognition ofach �-TrCP substrate, such molecules could provide
ore desirable intervention targets. The activation of
he IKK requires the formation of K63 type of ubiq-itin chains. Targets for K63 ubiquitylation are TRAF2,RAF6, RIP1 and the �-subunit of the IKK (Fig. 2).
lves among other ubiquitin-conjugating enzymes, TRAF2, TRAF6,of ubiquitin K63 chains and involves the Ubc13/UEV1 complex. The
s its degradation by the proteasome. The stop sign indicates putative
These modifications allow the recruitment and activa-tion of TAK1 through TAB1/TAB2 ubiquitin bindingproteins and conclude with the activation of the IKKcomplex (Kanayama et al., 2004). The formation ofthe CARMA1/BCL10/MALT1 complex by the TCR-mediated activation of PKC� (Thome, 2004) results inthe modification of the �-subunit of the IKK with K63ubiquitin type of chains by MALT1. This IKK subunitcan also be modified by TRAF6. In the NF-�B path-way all conjugations of poly-ubiquitin chains throughlysine 63 are catalyzed by a heterodimer formed by theubiquitin conjugating enzyme UBC13 and the UEV1protein (Fig. 2). A screening of chemical combinatoriallibraries based on yeast two-hybrid readouts have per-mitted the identification of small molecules that competewith UEV1 for its interaction with UBC13 and inhibit its
enzymatic activity (Thomson, personal communication).These compounds were shown to inhibit the activationof NF-�B by TNF-alpha, and also the formation ofK63-type poly-ubiquitin chains on another UBC13-UEV
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target protein, the DNA polymerase cofactor PCNA. Thepotential of these compounds as anti-cancer agents issupported by the fact that they sensitize tumour cellsto genotoxic agents, an activity that may be the con-sequence of the combined effects of these compoundsas inhibitors of NF-�B in response to diverse stim-uli, and as inhibitors of PCNA-mediated DNA damagetolerance. This is the first development of pharma-cological inhibitors of K63-mediated ubiquitin chainsand the first time that these compounds produce pos-itive effects and large potential to be used in futuretherapies. Another target for intervention in the NF-�B pathway is the tumour suppressor protease CYLD.This DUB appears to be mutated in familiar cylindro-matosis and is down-regulated in various cancer cells(Massoumi, Chmielarska, Hennecke, Pfeifer, & Fassler,2006; Strobel et al., 2002). CYLD removes K63 ubiqui-tin chains from TRAF2, interfering with the signallingcascade that activates the I�B kinase IKK and con-sequently affects the survival response mediated byNF-�B. Stabilising CYLD or promoting its activity maybe of benefit in cases with an aberrant NF-�B activitysuch as patients with inflammatory or immune disordersor Hodgkin’s disease (Karin, 2006).
5. Controlling subcellular distribution
Controlling subcellular distribution of a particularubiquitin-proteasome substrate might represent novelapproaches to prevent proteolysis, as changes in localiza-tion often condition proteasomal degradation (Salmena& Pandolfi, 2007). Examples include many transcrip-tion factors, such as E2F1 (Ivanova & Dagnino, 2007)and p53 (O’Keefe, Li, & Zhang, 2003) which requirenuclear export and nucleocytoplasmic shuttling for theirrespective proteasomal degradation. Shuttling of p53 ismediated by NES/NLS signals intrinsic to p53, or bya piggyback mechanism due to signals in Mdm2. Inaddition, Mdm2-mediated mono-ubiquitylation of p53in the nucleus leads to nuclear export (Li et al., 2003;Lohrum, Woods, Ludwig, Balint, & Vousden, 2001)(Fig. 3). The SENtrin-specific Protease 2 (SENP2) andthe p53 regulator MdmX (Mdm4) are additional exam-ples of factors whose localization is modulating theirproteasomal degradation. MdmX is highly homologousto Mdm2, but lacks ubiquitin ligase activity. MdmXheterodimerizes with Mdm2, and has dual roles inregulating Mdm2 function, enhancing and inhibiting
ubiquitylation of p53 and Rb, respectively (Badciong& Haas, 2002; Uchida et al., 2006). MdmX is ubiquity-lated by Mdm2, followed by proteasomal degradation.This degradation likely takes place in the nucleus, as
iochemistry & Cell Biology 40 (2008) 1126–1140
DNA damage stimuli leads to both nuclear translocationand turnover of MdmX (LeBron, Chen, Gilkes, & Chen,2006).
The multifunctional protein Jab1 induces the nuclearexport and cytoplasmic degradation of several differentproteins, such as p27Kip1, Smad4, Smad7 and p53 (Lee,Oh, & Song, 2006). Jab1 mediated nuclear export ofp53 depends on the nuclear export receptor CRM1 andon the preceding ubiquitylation of p53 by Mdm2 (Lee etal., 2006). The existence of adaptor proteins such as Jab1may provide a means of moderating the nuclear exportof particular proteins, in contrast to, e.g. the function ofthe toxin leptomycin B, which inhibits CRM1 dependentnuclear export of all nuclear proteins indiscriminately.Whether this strategy could prove to be a means of poten-tiating the functions of cellular factors, such as p53, byevading cytoplasmic degradation is unclear, as degrada-tion by nuclear proteasomes may provide an alternativeproteolytical route for nucleoplasmically accumulatedprotein (Renard et al., 2000).
The cytoplasmic ubiquitin ligase PARC can sequesterp53 in the cytoplasm, thus preventing p53 transcriptionalfunctions. This effect, however, is not dependent on theligase activity of PARC, as p53 cannot be ubiquitylatedby this particular E3 (Nikolaev, Li, Puskas, Qin, & Gu,2003) PARC shows increased expression levels in someneuroblastoma cell lines. These exhibit a cytoplasmicaccumulation of p53, corresponding to a heavily ubiq-uitylated form of p53 (Becker, Marchenko, Maurice, &Moll, 2007). Becker and colleagues argue that impair-ment of the interaction between the de-ubiquitylatingprotease USP7 and p53 may be responsible for thisaccumulation, as the binding sites on p53 for PARCand USP7 overlap. However, the massively ubiquity-lated and cytoplasmic p53 is still a proteasomal substrate.Thus, overexpression of PARC in cancer may lead toboth sequestration of p53 away from the nucleus, mas-sive ubiquitin modification and ultimately proteasomaldegradation. Interfering with the binding event betweenPARC and p53 may have several effects, such as restoredp53 de-ubiquitylation by USP7 and hence preventionof proteasomal degradation, restored p53 shuttling abil-ity, resulting in normal protein regulation, and abilityto accumulate in the nucleus to mediate transcriptionalfunctions. Similarly, the action of another DUB, CYLD,on the oncoprotein Bcl3, blocks its nuclear localisationand therefore survival mediated by NF-�B transcription(Massoumi et al., 2006). Tactics based on controlling
compartmentalization of substrates, prior to spatiallyregulated modification and degradation, may thus repre-sent an alternative approach for preventing proteasomaldegradation. The challenge here will probably lie in how
R. Hjerpe, M.S. Rodrıguez / The International Journal of Biochemistry & Cell Biology 40 (2008) 1126–1140 1135
Fig. 3. Ubiquitin-mediated regulation of p53 activity. Basal or low p53 activity promotes monoubiquitin-dependent cytoplasmic localisation of p53.USP7 mediated de-conjugation of ubiquitin has been proposed to promote nuclear import of p53. In the nucleus p53 will promote among others,transcription of genes involved in cell cycle progression and apoptosis. At early stages after a stress signal, p53 is transiently degraded by Mdm2a inactivat systemj erventio
ttd
taotudiHdcuPpEsMtc(
nd the proteasome. This signalling also favours the ARF-mediatedranscription dependent mechanisms will negatively regulate the p53ust before stimulation. The stop sign indicates potential points for int
o obtain specificity when interfering with compartmen-alization of a particular protein, as many factors likelyepend on similar mechanism for their trafficking.
In addition to substrate partitioning within the cell,he different parts of the ubiquitylation machinery arelso contained in different compartments as a meansf regulation. A family of E2’s has been reported toranslocate to the nucleus only after being loaded withbiquitin in the cytoplasm. This change of localization isependent on the karyopherin importin-11, which onlynteracts with the ubiquitin charged form of these E2’s.owever, authors mention that this interaction may beependent on specific adaptor proteins, such as UBDontaining factors, as interaction could not be shownsing bacterially purified recombinant proteins (Plafker,lafker, Weissman, & Macara, 2004). This hints to theossibility of restricting the movement of a subclass of2’s by precise interference with these adaptors, con-equently inhibiting the activity of their cognate E3’s.
any ubiquitin E3’s are also subject to spatial con-rol by being in, or travelling between, specific cellularompartments—examples include among others Nedd4Hamilton, Tcherepanova, Huibregtse, & McDonnell,
tion of Mdm2 resulting in a stabilisation and activation of p53. Theuntil it reaches the homeostatic balance similar to the one observedn.
2001; Shearwin-Whyatt, Dalton, Foot, & Kumar, 2006),VHL (Lee et al., 1999), the ER associated ligases Doa10(Deng & Hochstrasser, 2006) and Hrd1 (Hampton,2002), the BRCA1/BARD1 complex (Henderson, 2005)and Mdm2. While shuttling of Mdm2 is dispensablefor p53 degradation, as long as p53 itself can shuttle(O’Keefe et al., 2003), it cannot be excluded that ubiq-uitylation of other Mdm2 substrates may depend on itsmobility between nucleus and cytoplasm.
The ARF tumour suppressor can stabilize p53 by,among other processes, sequestering Mdm2 in the nucle-olus (Weber, Taylor, Roussel, Sherr, & Bar-Sagi, 1999).Additionally, ARF also stabilizes the Rb tumour suppres-sor by its interaction with Hdm2 (Chang et al., 2007).NPM is a nucleolar phosphoprotein, interacting withboth Mdm2 and ARF, and stabilizing p53 by binding toHdm2 in the nucleoplasm. The fact that ARF and NPMinteract with and stabilize each other has led to the sug-gestion that both proteins may cooperatively sequester
Hdm2 in the nucleolus. However, increased expressionof NPM has also been observed in cancer, and it hasbeen suggested that NPM may antagonize the tumoursuppressive effect of ARF (Gjerset, 2006; Gjerset &
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Bandyopadhyay, 2006). The possibility of potentiatingARF’s ability to hold Hdm2 in the nucleolus, throughinterference with ARF–NPM complex formation couldprove to be novel tactic to prevent the proteasomaldegradation of tumour suppressors such as Rb and p53.Moreover, PML can also bring Mdm2 to the nucleolus,and thus stabilize p53 (Bernardi et al., 2004). In cancercell lines such as HeLa and H1299, PML association tothe nucleolus is severely diminished. However, stimula-tion dependent re-association of PML to the nucleolusin HeLa cells is accompanied by induction of senescentphenotypes and increased protein levels of p53, pRB andp21 (Janderova-Rossmeislova et al., 2007).
It was recently shown that ubiquitin independentdegradation of p21Cip1, p16INK4a and ARF is controlledby a subset of proteasomes containing the REG� (PA28)proteasomal subunit. REG� exists as a heptameric com-plex (Wilk, Chen, & Magnusson, 2000), forming an 11Scap structure, gating access of substrates to the 20S coreparticle. As REG� has a nuclear restricted expressionpattern (Moriishi et al., 2003), this proteasomal compo-sition may degrade a specific set of substrates residingin the nucleus (Chen, Barton, Chi, Clurman, & Roberts,2007). Targeting a REG� function, such as heptamericassembly, may prove to be a strategy to inhibit the protea-somal degradation of cellular factors crucial for tumoursuppression, for example the ARF protein.
6. Tackling transcription
Gene transcription is up/down regulated by a widevariety of enzymes in the UPS. Accumulated evidenceindicates that since the assembly of transcriptionalpre-initiation complexes, promoter clearance, transcrip-tion elongation and until the export of mRNAs, manysteps are controlled by proteolytic and non-proteolyticmechanisms dependent on ubiquitylation (Gwizdek etal., 2005; Morris, 2004). The first evidence demon-strating the involvement of ubiquitin in initiation oftranscription came from Tansey and collaborators whoexpressed in yeast a fusion of ubiquitin and the arti-ficial activator LexA-VP16 to restore transcription ofthe LexA-responsive reporter gene in a strain deletedof the gene encoding the ubiquitin E3 ligase Met30(Salghetti, Caudy, Chenoweth, & Tansey, 2001). Fromthese experiments emerged the notion that mono-ubiquitylation of an activator promotes transcriptionalactivity, which was further developed by various groups
who reached the conclusion that extension of ubiqui-tin chains through K48 could terminate transcription bypromoting proteasome-mediated degradation (Kodadek,Sikder, & Nalley, 2006; Ryo et al., 2003; Tanaka, Grusby,
iochemistry & Cell Biology 40 (2008) 1126–1140
& Kaisho, 2007). Mono-ubiquitylation of the HIV-1transactivator protein Tat has also been shown to increasetranscription of the HIV-1 long terminal repeat (LTR)(Bres et al., 2003). In this case, ubiquitin-conjugation ismediated by the E3 ligase Hdm2, which modifies in addi-tion to p53 other cellular substrates such as the HistoneAcetyltransferase Tip60. Recently published evidenceindicates that the ubiquitin-ligase E4F1, mediate ubiq-uitylation of p53 through the formation of K48 chainspromoting transcription but not proteasome-mediateddegradation (Le Cam et al., 2006) (Fig. 3). Such an atyp-ical function of this type of poly-ubiquitin chains appearto be specific for a group of lysine residues on p53 (K319,K320 and K321) different to the ones involved in theproteasome-mediated degradation (Rodriguez, Desterro,Lain, Lane, & Hay, 2000), suggesting structural speci-ficity in this process.
Although the precise molecular mechanisms regu-lating the ubiquitin-mediated initiation and terminationof transcription remain elusive, accumulating evidencesupports a model where ubiquitylation of transcriptionfactors promote their transcriptional activity, proba-bly through the recruitment of general transcriptionalmachinery or co-activator complexes. Based on studiesof regulation of the estrogen receptor � (ER-�) pro-moter, the role of the proteasome in the disassemblyof transcription complexes was suggested (Reid et al.,2003). In this way cycles of association and dissocia-tion of functional transcriptional complexes coordinatedby ubiquitylation will occur. There is published dataindicating that this could be also the case for p53 andNF-�B (Saccani, Marazzi, Beg, & Natoli, 2004; Zhu etal., 2007). At present, we are far from having evidence tosupport the notion that ubiquitylation regulates all genepromoters whose expression responds to stimulation,in which case, understanding specificity will be criticalin discriminating gene targets for potential interventionstrategies.
7. Conclusions
Initially associated with protein degradation via the26S proteasome, the actual view of the function ofubiquitylation is changing. In addition to proteolysis,ubiquitylation should also be associated to several alter-native processes that may not be linked to proteindegradation. As many other post-translational modifi-cations, but in contrast those inducing small changes
in the charge of the protein, ubiquitylation leads todrastic changes in biochemical properties of the sub-strate, resulting in changes of protein function. Dueto its characteristic high reversibility, many functions
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R. Hjerpe, M.S. Rodrıguez / The International Jour
ay be modulated just by acting on the activity of theonjugating or de-conjugating enzymes. In cases like53, multiple types of ubiquitin chains participate inhe regulation of the various activities of the target pro-ein (Fig. 3). Extensively studied protein models suggesthat after the accomplishment of a ubiquitin-dependentunction, ubiquitylation appears to guarantee the termi-ation step by promoting protein degradation and drawspicture of proteolysis merely being the consequence
f a process already achieved that requires the exis-ence of cyclic up/down regulated processes to keep cellslive (Fig. 3). Independently of the function, many vitalactors are tightly regulated with a very delicate equilib-ium between enhanced/repressed activity mechanisms.his homeostatic balance is controlled by negative andositive feedback loops that will tend to re-equilibratehe system as soon as the physiological or pathologicalvent has been overcome. Breaking down this dynamicalance by promoting an exacerbated positive or nega-ive response might have dramatic consequences for theell. The UPS actively participates in the regulation ofany signal-induced processes and establishes a molec-
lar symbiosis with co-factors regulating the activity ofhe target protein. When the molecular symbiosis exist-ng between the UPS and regulated cellular factors iserturbed this results in aberrations that are typicallybserved in a large variety of pathologies which includeeurodegenerative disorders and cancer.
The challenge for new inhibitors tackling targetsn the UPS, upstream of the 26S proteasome, will beot only to regulate protein stability to extend pro-ein function over time, but also to contribute to locateroteins in the right compartment, accessible to otherost-translational modifications and to promote associa-ions with critical regulators. To avoid side effects and toimit doses of inhibitors applied, specific protein–proteinnteractions should be considered as targets for drugevelopment, rather than general functions, such as ubiq-itylation mediated by enzymes involved in multiplerocesses or proteasome-mediated degradation. The facthat all components of the ubiquitylation machineryxhibit some form of spatial regulation is indicative ofhe importance of subcellular localization in the mod-fication of substrates. Helping the cell to find its owne-equilibrium using internal tools and mechanisms,ould be a strategy that might generate less undesiredffects and might be of much interest in chronic patholo-ies such as immune and inflammatory diseases. Taking
nto consideration not only prolonged protein half-life,hich not necessarily will correspond to prolonged pro-
ein function, but also how inhibition of degradation willffect transcriptional capacities, subcellular partition-
ing, post-translational modification and de-modification,interaction with critical partners, etc., will certainly beimportant for future approaches directed to prevent theproteasomal degradation of therapeutical targets.
Acknowledgements
We thank the Ubiquitin-Like Proteins and Cancergroup for motivating discussions considered in thisreview. Thanks to Valerie Lang, Christine Blattner, MayMorris and Timothy Thomson, members of the INPRO-TEOLYS network and David Hay and Jim Sutherlandfor the critical reading of this manuscript. Our group issupported by the Ramon y Cajal Program, Ministeriode Educacion y Ciencia grant BFU 2005-04091, Fondode Investigaciones Sanitarias (FIS) CIBERhed, Depart-ment of Industry, Tourism and Trade of the Governmentof the Autonomous Community of the Basque Country(Etortek Research Programs) and from the InnovationTechnology Department of the Bizkaia County.
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