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Leukemia Research 34 (2010) 1403–1409

Contents lists available at ScienceDirect

Leukemia Research

journa l homepage: www.e lsev ier .com/ locate / leukres

nvited review

ethods for measuring proteasome activity: Current limitations anduture developments

. Liggetta,b,c, L.J. Crawforda,b,c, B. Walkera,b,c, T.C.M. Morrisa,b,c, A.E. Irvinea,b,c,∗

Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, Belfast, UKDepartment of Pharmacy, Queen’s University, Belfast, Belfast, UKDepartment of Haematology, Belfast Health and Social Care Trust, Belfast, UK

r t i c l e i n f o

rticle history:eceived 25 February 2010

a b s t r a c t

The proteasome has been validated as a therapeutic target, with proteasome inhibitors showing particularefficacy in the treatment of Multiple Myeloma. A wide range of methods have been developed to profile

eceived in revised form 10 June 2010ccepted 3 July 2010vailable online 31 July 2010

eywords:roteasome

proteasome activity. These include the current method of choice fluorogenic peptide substrates, as wellas bioluminescent imaging, immunological methods, and more recently, site-specific fluorescent probes.The aim of this review is to evaluate the currently available methods for profiling proteasome activityand their suitability for use in translational studies. Ongoing development of techniques for profilingproteasome activity will facilitate future research into proteasome-related pathologies, thus accelerating

luorogenic peptide substratesssay

the development of more specific drug regimes.© 2010 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14032. Proteasome preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14053. Fluorogenic peptide substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1405

3.1. Proteasome activity in crude cell extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14053.2. Proteasome activity in plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14053.3. Proteasome activity in whole cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1405

4. Site-specific activity probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14065. Two-step activity based protein profiling (ABPP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14066. Bioluminescent imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14067. RFDD-PCR and proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14078. Immunological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14079. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1408

Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1408Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1408References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1408

. Introduction

The degradation of cellular proteins is a highly complexnd tightly regulated process that plays an important role in

∗ Corresponding author at: Centre for Cancer Research and Cell Biology, Queen’sniversity Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK.el.: +44 28 90972794; fax: +44 28 90972776.

E-mail address: s.irvine@qub.ac.uk (A.E. Irvine).

145-2126/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2010.07.003

regulating cellular function and maintaining homeostasis. Theubiquitin–proteasome system (UPS) is the major pathway for intra-cellular protein degradation. The UPS is made up of two main,highly regulated, steps. The first is the tagging of the target proteinwith a polyubiquitin chain and the second is the subsequent degra-dation of the protein by the 20S catalytic core of the proteasome [1].

Proteins are tagged with ubiquitin (Ub) via a step-wise mechanism[2] involving three enzymes: E1 (Ub activating enzyme), E2 (Ubconjugating enzyme) and E3 (Ub ligase). 26S proteasome recog-nises and binds the polyubiquitinated protein complex and thisis subsequently unfolded and fed into the 20S catalytic core. The

1404 A. Liggett et al. / Leukemia Research 34 (2010) 1403–1409

Fig. 1. Outline of the ubiquitin–proteasome system. An E1 ubiquitin-activating enzyme binds ubiquitin (Ub); ubiquitin is transferred to an E2 ubiquitin-conjugating enzyme;a sfer ofa asomei

Uc

elsoih(traiysp

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Fl

n E3 ubiquitin ligase subsequently recruits the target protein and mediates the tranpolyubiquitin chain that functions as a signal to target the protein to the 26S prote

s recycled.

b molecules are recycled and the protein is broken down into itsonstituent amino acids (Fig. 1).

The 26S proteasome is a multicatalytic complex found in allukaryotic cells. It is composed of a 20S core with one or two regu-atory 19S subunits attached (Fig. 2). The 20S core is a barrel-shapedtructure made up of two inner rings of seven � subunits and twouter rings of seven � subunits [3]. The three main catalytic activ-ties associated with the proteasome are located within the twoeptameric � rings. These are referred to as: the chymotrypsin-likeCT-L) activity (�5 subunit), since it cleaves after acidic residues, therypsin-like (T-L) activity (�2 subunit), which cleaves after basicesidues and the peptidylglutamate peptide hydrolysing (PGPH)ctivity (�1 subunit), which is also referred to as the caspase activ-ty due to its affinity to cleave after aspartic residues. Studies ineast and bovine liver have shown that even between species, theame 14 subunits are present in 20S proteasomes, verifying that theroteasome is a uniform, highly ordered enzymatic complex [4].

In mammalian cells, an alternate form of the proteasome,eferred to as the immunoproteasome, is generated in response

o interferon gamma (IFN�). This is composed of a central 20Sore and one or two 11S regulatory particles, also known asA28. The three active site � subunits, �1, �2 and �5 areeplaced by �1i (RING12/LMP2), �2i (MECL-1) and �5i (C1/LMP7),espectively. These cleave substrates at different amino acid

ig. 2. The 26S proteasome is composed of a 20S catalytic core and two 19S regu-atory caps.

ubiquitin to the protein. The successive conjugation of ubiquitin moieties generatesfor degradation. Targeted proteins are broken down to oligopeptides and ubiquitin

residues to their regular counterparts. This alters the proteolyticspecificity to favour the generation of antigenic peptides forpresentation by major histocompatibility complex (MHC) class Imolecules. The immunoproteasome is expressed mainly in lym-phoid tissues such as the spleen, lymph nodes and thymus[5].

The proteasome is involved in the regulation of a number ofvital cellular processes including cell cycle regulation, differentia-tion and apoptosis. It is therefore not surprising that it has beenimplicated in the pathogenesis of a number of diseases includingAlzheimers Disease [6,7], ischaemic stroke [8], diabetes [9] and avariety of cancers [10,11]. In the past decade, proteasome inhibitorshave been highlighted as potential therapeutic agents. The pro-teasome inhibitor, Velcade® (Bortezomib, PS-341) was the firstclinically available proteasome inhibitor and was approved by theFDA in 2003 for the treatment of Multiple Myeloma [12]. Clinicalstudies with Velcade validated the proteasome as a legitimate ther-apeutic target and following on from its success, a number of novelproteasome inhibitors were developed. Two of these compounds,NPI-0052 and Carfilzomib, with distinct chemical structures andaffinity for the proteasome subunits are currently undergoing clin-ical trials.

As the essential role of the UPS in cellular function became evi-dent and proteasome inhibitors came to the fore as therapeuticagents, numerous assays to profile and characterise the proteasomehave been developed. One limitation of many conventional meth-ods to measure proteasome activity is the number of cells required,which can render them unsuitable for use in clinical studies. Withthe continual development of novel proteasome inhibitors and theincrease in their use, there is a real need for the development ofnovel methods to profile proteasome activity in clinically relevantnumbers of cells. This would allow us to predict which patients

might benefit from this therapeutic strategy and monitor responseto treatment. The aim of this review is to describe the currentlyavailable methods for assessing proteasome activity and evaluatetheir use and suitability for application to future clinical investiga-tions.

A. Liggett et al. / Leukemia Resea

Table 1Fluorogenic peptide substrates to measure 26S proteasome activity.

Proteasomal catalytic subunit Fluorogenic peptide substrate

�5, Chymotrypsin-like (CT-L) Suc-LLVY-AMCZ-GGL-AMCSucc-AAF-AMC

�2, Trypsin-like (T-L) Boc-LRR-AMCZ-ARR-AMCBz-FVR-AMCBz-VGR-AMCBoc-LSTR-AMCAc-RLR-AMC

�1, Post-glutamate peptide Z-LLE-NA

2

ei2irgOu(stcstcpteoM

3

atapibflflcco2mha

3

thd

hydrolysing (PGPH) Z-LLE-AMCAc-nLPnLD-AMCAc-GPLD-AMC

. Proteasome preparations

A number of methods have been developed to prepare cellxtracts containing 20S and 26S proteasomes. These differ depend-ng on both proteasome type and scale of purification. Since0S proteasomes are widely abundant in eukaryotic cells, mak-

ng up 0.5–1.0% of total cellular protein, no special technique isequired [13]. For small-scale proteasome preparations, a densityradient centrifugation using 10–40% glycerol is sufficient [13].ptimal small-scale extraction of 20S proteasomes is achievedsing an ATP/DTT cell lysis buffer consisting of 10 mM Tris–HClpH 7.8), 0.5 mM DTT, 5 mM ATP and 5 mM MgCl2 [14]. For large-cale preparations, all buffers contain 10–20% glycerol to keephe activity stable. For 26S proteasome preparations, all buffersontain 20% glycerol together with 2 mM ATP and 1 mM DTT fortabilization [13]. Further options include the use of differen-ial centrifugation together with gel filtration and anion-exchangehromatography to purify 20S and 26S proteasomes [15]. Theseroteasome subtypes can be further separated into their consti-utive subunits by the use of affinity chromatography or native gellectrophoresis [15–17] and identified by matrix-assisted laser des-rption ionization time-of-flight mass spectrometry (MALDI-TOFS) [16,17].

. Fluorogenic peptide substrates

The most commonly cited method for profiling proteasomectivity is the use of fluorescently tagged substrates specific for thehree main components of proteasome activity. These have beenpplied to monitoring proteasome activity in crude cell extracts,lasma and whole cells. The fluorescence is initially quenched in the

ntact molecule, and released following cleavage of the substratey the specific proteasomal active site. Therefore, the increase inuorescence is proportional to proteasome activity. A number ofuorogenic peptides have been designed to profile the three mainatalytic activities of the proteasome (Table 1). Each of these areomposed of three or four amino acid substrates attached to a flu-rescent tag, most commonly, 7-amino-4-methylcoumarin (AMC),-naphtylamine (NA), 4-methoxy-2-naphtylamine (MNA) [18], orethyl-coumaryl-7-amide (MCA) [13]. Of these, AMC exhibits the

ighest fluorescence and is most widely used to profile proteasomalctivity.

.1. Proteasome activity in crude cell extracts

Fluorogenic peptide substrates are routinely used to monitor thehree enzymatic activities of 26S proteasomes in crude cell extracts,owever, their non-specificity in this application has been readilyocumented and needs to be considered when using this method

rch 34 (2010) 1403–1409 1405

[18–21]. Reidlinger et al. described the non-specificity of fluoro-genic substrates for assessing 20S activity in crude cell extractssince other proteins present in the extracts can inhibit the hydrol-ysis of Suc-LLVY-AMC by 20S but not 26S proteasomes [19]. Theynoted that the presence of casein inhibited hydrolysis of Suc-LLVY-AMC by 20S proteasomes, but slightly stimulated hydrolysis by 26Sproteasomes.

Rivett et al. used a number of fluorescently tagged substrates tocreate assays that could monitor changes in total cellular protea-some activity in crude cell extracts and determine the contributionof the various purified intracellular proteasome complexes, 26Sproteasomes, 20S proteasomes and PA28-20S proteasome [20].This study was carried out using both crude cell extracts and puri-fied 20S proteasomes, 26S proteasomes and the regulatory 11Ssubunit, PA28, from rat liver preparations. Rivett et al. noted a num-ber of problems with this method. The activity measured usingcrude cell extracts comprises all of the component activities ofproteasome subunits and complexes. Although distinct activitypatterns were observed between purified complexes, for examplethe 26S exhibited much higher activity than the 20S core, the actualactivities from each were difficult to quantify, since these dependedon the substrate used. The observed activities were also affected bythe proportion of each complex present in the cell lysate. This variesbetween tissues and cell types and was estimated by separationof each of the proteasome sub-complexes, achieved by gel filtra-tion or glycerol gradient centrifugation, and determination of theratio of activities. These studies concluded that this method is use-ful for assaying changes in total cellular proteasome activity fromcrude cell extracts. The non-specificity limits the application of thismethod to individual, purified proteasome complexes since 26Sproteasomal activity could only be accurately measured followingpurification to remove additional cellular proteases, competitorsfor the respective catalytic sites and any potential activators orinhibitors of the proteasome.

3.2. Proteasome activity in plasma

Ma et al. have recently used the fluorogenic substrates Suc-LLVY-AMC, BZ-VGR-AMC and Z-LLE-AMC to study the CT-L, T-L andPGPH components of proteasome activity, respectively, in circulat-ing proteasomes in patients suffering from Chronic LymphocyticLeukaemia (CLL) [22]. This method may prove useful in future clin-ical studies since they reported that the enzymatic activities incirculating proteasomes are a useful marker in patient monitoringand disease prognosis in CLL.

3.3. Proteasome activity in whole cells

In recent years, conventional fluorogenic substrates have beenadapted for measuring proteasome activity directly in whole cells.A study by Elliott used the conventional fluorogenic substrates Suc-LLVY-AMC and Bz-VGR-AMC to profile the CT-L and T-L proteasomeactivities respectively in whole blood samples or sub-populationsof blood cells [23]. In this investigation, peripheral blood was sepa-rated and the selected cells were lysed, incubated with fluorescentsubstrate and proteasome activity was recorded as an increase influorescence. Significant differences were recorded in proteasomeactivity between individuals, however, there were non-significantdifferences in activity within individuals. These assays showedpotential for monitoring real-time pharmacodynamics in patientstreated with Velcade, however there has been no recent evidence

of their development for use in a clinical setting.

In general, the application of fluorescent peptide substrates toclinical investigations is limited due to their lack of specificity.However, these substrates can still provide a useful tool for in vitrocharacterisation of the proteasome and for initial screens to char-

1406 A. Liggett et al. / Leukemia Research 34 (2010) 1403–1409

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ig. 3. Fluorescent proteasome probes can be used to assess proteasome activity in

cterise the potency and effect of novel inhibitors on the individualubunit activities.

. Site-specific activity probes

Active site-directed probes have emerged as improved tools foronitoring proteasome activity. They show promise for applica-

ion to clinical research both in the characterisation of proteasomectivity in clinical samples and in the characterisation of novelroteasome inhibitors (Fig. 3) [24–26]. Active site-directed probesre cell-permeable peptide vinyl sulfone proteasome inhibitorshat have been modified to function as active site-directed probesf the proteasome. Initially these compounds were synthesisedith a radio-isotope or azide group attached, however, these

abels made the compounds impermeable to cells. Addition of amall hapten, dansyl-sulfonamidohexanoyl tag to these inhibitorsllowed them to retain cell permeability and irreversibly bindo the exposed catalytic subunits of both the proteasome andmmunoproteasome in living cells. Antibodies directed againsthe dansyl-sulfonamidohexanoyl hapten can be used to assayroteasome activity and characterise proteasome inhibitors by

mmunoblotting [24]. More recently, this probe has been fur-her optimized by replacing the dansyl tag with high-quantumuorophores. This modification allows direct measurement of pro-easome activity using in-gel profiling, confocal microscopy andow cytometry [25]. Additionally, Hasegawa et al. have describednovel activity based probe to profile proteasome activity in wholeells based on belactosin A, a potent proteasome inhibitor derivedrom Streptomyces metabolite [26]. The probe, dansyl-KF33955,ovalently and specifically binds to and labels the catalytic activeites of the 26S proteasome in whole cells in vivo.

Our group and others have employed these probes to profile thectivities of the constitutive and immunoproteasome in a varietyf haematological malignancies and have shown that the activityatterns of individual proteasome subunits are variable betweenisease types [27,28]. Kraus et al. went on to demonstrate an upreg-lation of active subunits in Velcade-resistant cells and hypothesizehat the relative contribution of CT-L and T-L activities may influ-nce the degree of Velcade sensitivity [28].

. Two-step activity based protein profiling (ABPP)

Activity based protein profiling (ABPP) involves the applicationf chemical probes to monitor protein dynamics across complex

cells by gel-based assays, by confocal laser scanning and by flow cytometry [27].

proteomes. Verdoes et al. together with Cravatt et al. have reportedthe potential for two-step activity based protein profiling for usewith the proteasome and serine hydrolases [29,30]. Initially, cellsare incubated with an activity based probe (ABP), modified to con-tain a small biocompatible reactive group such as an azide oracetylene. The ABP interacts irreversibly and covalently with thespecific target enzyme, and a reporter group is introduced in achemoselective manner, either by Staudinger–Bertozzi ligation orHuisgen cycloaddition. Staudinger–Bertozzi ligation is a chemicalreaction in which an azide is combined with a phosphine or phos-phate and subsequently reduced to an amine [31]. The Huisgenreaction is an organic chemical reaction which takes place in thepresence or absence of copper (I) and involves the cycloadditionof an azide to a terminal alkyne to form a 1, 2, 3-triazole [32,33].Verdoes et al. have demonstrated the application of a novel bifunc-tional fluorophore, azido-BODIPY acid 1 to two-step ABPP of totalcellular proteasome activity in both cell lysates and whole cellpreparations. This method requires 2 × 106 cells per experimentand additional Western blotting techniques to profile activities.

ABPP shows potential for monitoring efficacy and patientresponse to proteasome inhibitors in a variety of disease statessince it overcomes the problems associated with conventional flu-orogenic substrates such as cell permeability and bioavailability,which will aid studies of proteasome activity in animal models.

6. Bioluminescent imaging

Molecular imaging is defined as the measurement of biologicalprocesses at the cellular and molecular level using remote imagingdetectors. It is a non-invasive process, which focuses on monitor-ing gene expression in vivo. Together with the information nowknown from the map of the human genome and the recent surgeof research into gene function, molecular imaging has the potentialfor involvement with both basic and translational research as wellas patient care.

Two approaches to measuring proteasome activity using biolu-minescent imaging have been considered:

(1) The use of exogenously delivered probes that are subsequentlyactivated or de-activated following proteasomal processing.

(2) The use of genetically encoded reporters, which act as protea-some substrates.

A. Liggett et al. / Leukemia Research 34 (2010) 1403–1409 1407

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ig. 4. Basis of the bioluminescent proteasome assay. Proteasome substrates Suc-LLminoluciferin, which is used by luciferase to generate light. The amount of light ge

In each case, the level of activity of the proteasome towardshose substrates can be monitored as a direct positive correlationf the signal produced. Ubiquitin firefly luciferase (Ub-FL) has beenesigned to monitor total proteasomal activity [34,35] togetherith Ubiquitin-Green Fluorescent Protein (UbG76V-GFP) [34].

Moravec et al. have described new bioluminescent cell-basedssays that can be used to measure the three main proteasomectivities individually [36]. The basis of these assays is the lumino-enic proteasome substrates Suc-LLVY, Z-LRR and Z-nLPnLD for theT-L, T-L and PGPH activities, respectively (Fig. 4). Each substrate isissolved in buffer optimized for cell permeabilization, proteasomectivity and luciferase activity. Proteasome activity can be profiledn a minimum of 5 × 103 cultured cells in a single-step, multiwellormat before and after treatment with proteasome inhibitors. Theubstrate interacts with its specific proteasomal subunit, releas-ng aminoluciferin, which is used by luciferase to generate light,irectly proportional to proteasome activity. This method can besed in clinically relevant numbers of whole cells and eliminateshe need for cell lysates, reducing the required time and materials.

. RFDD-PCR and proteomics

Restriction fragment differential display polymerase chaineaction (RFDD-PCR) and proteomics have been applied toroteasome-related diseases to respectively identify differentiallyxpressed genes and study the structure and function of relatedroteins. Deng et al. carried out a study to determine the differ-nces in the gene expression of various proteasome subunits (PSs)nd ubiquitin specific peptidases (USPs) between normal tissue andreast cancer [37]. RFDD-PCR identified expressions of genes of PSs

nd USPs, depending on the number of base pairs, using a databaserovided by Qbio-gene Inc. and MP Biomedicals Inc. The expressionf these proteins was further investigated using two-dimensionalolyacrylamide gel electrophoresis (2-DE) and MALDI-TOF-TOF-S, with further verification using immunohistochemical staining.

RRR and Z-nLPnLD interact with the �5, �2 and �1 subunits respectively, releasinged is directly proportional to the activity of the proteasomal subunits [42].

This study highlighted 4 PSs and 5 USPs that may be potential tar-gets for the treatment of breast cancer and provide markers for theevaluation of patient prognosis.

In the study by Deng and colleagues, the over-expressed genesdetected by RFDD-PCR were inconsistent with over-expressed pro-teins observed with 2-DE and MALDI-TOF-TOF-MS. This may be dueto limited sensitivity of detection of corresponding over-expressedproteins, or some PSs and USPs may only function at the tran-scriptional level. The basis of this method holds promise for theidentification of important targets for treatments and monitoringof pathologies associated with the dysregulation of the proteasome.Further refinement of this approach is still required to improve itsreproducibility and sensitivity.

8. Immunological methods

Enzyme-linked Immunosorbent Assays (ELISA) have been usedto quantify the 20S core particle of the proteasome in humanplasma and serum for a number of years. Wada et al. were the firstgroup to detect and quantify 20S proteasomes in human serumusing this method [38]. They found a positive correlation betweenthe levels of circulating proteasomes and tumour burden of patientssuffering from haematological malignancies such as Non-Hodgkin’lymphoma and Multiple Myeloma. This was further investigated byLavabre-Bertrand et al. in a variety of haematological malignanciesand solid tumours, highlighting the levels of circulating protea-somes as a potential marker of disease progression [39]. However,the application was limited to purified 20S proteasomes in thesestudies, highlighting the need for further development to quantify26S proteasomes in crude cell extracts.

Majetschak and Sorell [40] have modified the 20S proteasomesandwich ELISA for use with cell extracts and crude lysates [38].They have also utilized the specific Mg2+/ATP requirement for 26Sproteasome formation and stability to develop the first sandwichELISA protocol for the 26S complex, showing no cross-talk with the

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408 A. Liggett et al. / Leukemia

0S complex. One weakness of this assay is its inability to distin-uish between single or double-capped 20S complexes. However,he protocols are easy to follow and reagents are commerciallyvailable, allowing them to be carried out in any routine laboratory.dditionally, during the development of these assays, Majetschaknd Sorell [40] observed the capacity of solid phase affinity immo-ilization (SPAI) of 26S proteasomes to characterise its dissociation

nto its constituent 19S and 20S particles, thus providing a new toolo investigate proteasome characteristics, with prospective appli-ation to clinical investigations.

. Conclusion

The proteasome is a multicatalytic complex found in all eukary-tic cells. Its dysregulation is associated in the pathogenesis of aumber of disease states including Alzheimers Disease and cancer.he proteasome has recently emerged as a potential therapeuticarget, with proteasome inhibitors showing particular efficacy in

ultiple Myeloma. A number of methods have been developed toonitor proteasomal activity. In the past these have been limited

o the use of purified proteasomes from cell lysates or serum. Theseethods often require high cell numbers, therefore rendering them

nsuitable for use with clinical samples where cell numbers areimited. In addition, the purification process may cause activationf the 20S subunit or affect 26S proteasome concentration and com-osition, therefore experiments carried out under these conditionsay not provide a true estimate of cellular proteasome function.

tudies have shown differences in the relative contribution of eachf the three main components of proteasome activity, CT-L, T-L andGPH, in purified proteasomes compared with crude cell extracts.his makes it difficult to assess the effects of treatment with agentsuch as Velcade on individual proteasomal subunits in living cells,hus highlighting the need for the development of more specific

ethods and improved tools to accurately measure the compo-ents of proteasomal activity in vivo.

Methods have recently been developed, which provide new andmproved ways of monitoring proteasome activity [24,25,28,41].hese novel approaches eliminate the need for preparation of cellysates and can be used directly with whole cell preparations.owever, many are still limited with regards to their application

o clinical material. The single-step homogenous bioluminescentssay developed by Moravec et al. shows great promise for futurelinical studies, since it can be used with a small number of cells36].

To date, the method of choice for the majority of investigatorss the use of the fluorogenic peptide substrates, however, with thexception of the study by Elliott et al., these have been shown toe limited to in vitro characterisation of the proteasome and pre-linical investigations of novel proteasome inhibitors [23]. With theontinual development and modification of methods to profile pro-easome activity, more information will be discovered about thisnzymatic complex in vivo. This will provide us with the neces-ary tools to fully investigate proteasome-related pathologies andead to the development of future, more specific, drug treatmentegimes.

onflicts of interest

Authors have no conflicts of interest.

cknowledgements

AL was funded by the Department of Education and Learning,orthern Ireland.

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rch 34 (2010) 1403–1409

Contributions: AL: manuscript writing; LC: manuscript writingand editing; BW and TM: final approval of manuscript and AI: edit-ing and final approval of manuscript.

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