WO2016034895A1 - Method and systems for investigating the ubiquitin-proteasome system - Google Patents

Abstract

A method is provided for investigating molecular interactions and enzyme activities related to the ubiquitin-proteasome system (UPS), the method comprising: a) providing at least one surface with a target moiety bound to at least a portion thereof; b) i) exposing said surface to a medium comprising a labelled ubiquitin moiety such as ubiquitin, SUMO, NEDD8 e.t.c., and a component or components of the ubiquitination system suitable to conjugate the ubiquitin moiety to a target moiety; and/or ii) wherein said target moiety is conjugated to a labelled ubiquitin moiety exposing said at least one surface to a medium comprising a deubiquitinating enzyme (DUB) suitable to remove the ubiquitin moiety from a target moiety; c) providing conditions permissive for the conjugation/removal of the labelled ubiquitin moiety to/from the target moiety; and d) detecting conjugation and/or removal of the labelled ubiquitin moiety by detecting labelled ubiquitin moiety located at or near the surface.

Description

METHOD AND SYSTEMS FOR INVESTIGATING THE

BIQUITIN-PROTEASOME SYSTEM

The present invention relates to methods and systems for the investigation of the ubiquitin- proteasome system (UPS). In particular, the invention relates to assays, methods and systems for investigation of UPS-related interactions, enzymatic activities and identification of agents which are able to modulate the UPS system.

Background of the Invention Ubiquitination comprises a series of reactions, which lead to the attachment of a ubiquitin protein to another protein, called a substrate. The ubiquitination cascade starts with ubiquitin activation by a ubiquitin-activating enzyme (E1), which forms a high energy thioester linkage with the C-terminus of ubiquitin in the presence of ATP as the energy source. E1 interacts with a ubiquitin-conjugating enzyme (E2) and ubiquitin is transferred to the E2 via a trans- thioesterification reaction. Finally, with the help of a ubiquitin ligase (E3), ubiquitin is typically attached to a lysine residue on a protein substrate, forming an isopeptide bond via its C- terminus (Figure 1). Depending on the type of modification (mono-, poly-, or multi- ubiquitination), the modified substrate protein can be targeted for proteasomal degradation, or can change its cellular localisation, interaction partners or enzymatic function.

Various classes of enzymes are involved in the ubiquitination process, leading to conjugation of ubiquitin to the substrate protein. The classical set includes the ubiquitin-activating enzyme E1 , ubiquitin-conjugating enzyme E2 and a ubiquitin E3 ligase. The ubiquitin transfer reaction can be represented as a pyramidal cascade, where two known human ubiquitin E1s transfer ubiquitin to one of dozens of E2s, which then interact with one or several of the hundreds of so far discovered E3 ligases, to finally specifically ubiquitinate thousands of known target proteins. To date, over 1200 core enzymes and ancillary proteins interconnected by around 50,000 interactions have been identified in the human ubiquitin system (Chatr-Arya mont et al., 2013). Through sequential repetitions of the ubiquitination process, other ubiquitin moieties can be attached to already conjugated ones, forming a polyubiquitin chain on the substrate. On the other hand, the deubiquitinating enzymes (DUBs), direct the opposite reaction of deubiquitination of substrates. Ubiquitin is not the only protein that can be conjugated to a substrate as numerous other ubiquitin-like (UBL) proteins are conjugated by analogous enzyme cascades. For simplicity, the term

"ubiquitination" will be used herein to describe ubiquitination and ubiquitin-like modifications in general, unless specified otherwise. Because of its complexity and multi-functionality, the ubiquitin-proteasome system (UPS) controls a vast range of cellular processes, including protein degradation, DNA repair and replication, cell cycle, secretion, transcription, translation and apoptosis, amongst others. Consequently, any deregulation in the system may strongly perturb cell function and result in disease, such as cancer, neurodegeneration, immune disorders, metabolic disorders, or cardiovascular disease (Petroski 2008). Therefore, the UPS is of great interest in terms of drug discovery. Two proteasome inhibitors, bortezomib and carfilzomib (Teicher et al., 1999; Kuhn et al., 2007), have now been approved by the FDA for multiple myeloma treatment. However, only a limited number of compounds that interfere with ubiquitination reactions have been reported so far due to an insufficient understanding of individual connections within the network, and the functionality and specificity of all the enzymes involved.

Indeed, despite an increasing number of high-throughput efforts aimed at understanding the UPS, around two-thirds of the system is still poorly characterised. Clearly, there is a need for novel high-throughput approaches to interrogate the UPS in a systematic manner which would form the basis for discovery of chemical modulators of specific ubiquitination reactions.

The present invention relates to an innovative, miniaturised method for interrogation of the UPS in vitro using confocal scanning technology (Hintersteiner et al., 2009, 2010, 2012; Meisner et al., 2009). The assay is designed to monitor in vitro ubiquitin conjugation to protein substrates in real-time and in a high-throughput way. It facilitates the measurement of ubiquitination conjugation dynamics, investigation of ubiquitination sites, and the activity of ubiquitinating enzymes. The assay applications include biochemical investigations of interactions and activities within the ubiquitination pathway and screening for inhibitors or enhancers of ubiquitination reactions.

Statements of the Invention In a first aspect the present invention provides a method for investigating molecular interactions and enzyme activities related to the ubiquitin-proteasome system (UPS), the method comprising:

a) providing at least one surface with a target moiety bound to at least a portion thereof; b) i) exposing said surface to a medium comprising a labelled ubiquitin moiety and a

component or components of the ubiquitination system suitable to conjugate the ubiquitin moiety to a target moiety; and/or ii) wherein said target moiety is conjugated to a labelled ubiquitin moiety exposing said at least one surface to a medium comprising a deubiquitinating enzyme (DUB) suitable to remove the ubiquitin moiety from a target moiety;

c) providing conditions permissive for the conjugation/removal of the labelled ubiquitin

moiety to/from the target moiety; and

d) detecting conjugation and/or removal of the labelled ubiquitin moiety by detecting

labelled ubiquitin moiety located at or near the surface.

The present method thus allows interrogation of ubiquitination and/or de-ubiquitination pathways (as set out in step b), parts i) and ii)).

The surface can be a surface of any suitable substrate. In some embodiments the substrate may comprise a matrix and the surface of the substrate may be the surface of the matrix. The surface of the matrix may correspond to a mesh of layers of cross-linking polymers, for example, and the target moiety may bind to one of the layers of the surface.

The substrate preferably comprises one or more particles and the surface can suitably be the outer surface of the particles. The particle(s) can be of any suitable size, but are suitably sized such that a plurality of particles can be provided in a single reaction vessel, e.g. the well of a plate; preferred sizes are discussed below. The particles can be any suitable shape, and mention can be made of spherical, spheroidal, polyhedral, plate-like (e.g. disc shaped or rectangular plates) or irregular shapes. Spherical beads are preferred in certain embodiments for a number of reasons, e.g. they are amenable to 'bead picking', and have a highly regular geometry which can be useful for handling and quantitative analysis.

Accordingly, in preferred embodiments the surface can be the outer surface of one or more beads, and various preferred feature of this embodiment are described in detail below.

Beads are typically substantially spherical, but other shapes of beads can be used, as discussed below. However, it should be noted the surface can potentially be any suitable surface to which a target moiety can be attached. For example, the surface can be a wall of a reaction vessel, e.g. the base or a side of a well provides a convenient surface. Accordingly, though it is typically less preferred, the substrate can be non-particulate, e.g. the wall of a reaction vessel or the like.

The term 'ubiquitin moiety' is used in the present application to cover ubiquitin and ubiquitin related modifiers (UBLs), including the 17 currently known human ubiquitin related modifiers in the classes NEDD8, SUMO, ISG15, FUB1 , FAT 10, Atg8, Atg12, Urm1 , and UFM1. It is possible that further ubiquitin-like moieties or unrelated protein modifiers will be identified, and such newly discovered moieties could of course be used in the present invention. The term 'ubiquitin moiety' is also intended to cover ubiquitin and ubiquitin related modifiers from other eukaryotes and ubiquitin-like proteins from prokaryotes. Furthermore, the term

'ubiquitin moiety' is intended to cover modified and mutated forms of ubiquitin or ubiquitin related modifiers, e.g. where the sequence has been modified by genetic engineering techniques. Preferred forms of the invention use mammalian ubiquitin moieties. For the avoidance of doubt, the term "conjugate the ubiquitin moiety to the target moiety" refers to the binding of a ubiquitin moiety to a target moiety via a chemical bond, such as via a covalent or non-covalent interaction.

The method is preferably used for detecting conjugation of a ubiquitin moiety to a target moiety. For example, the method can be used for detecting the addition of ubiquitin to a substrate protein or to a component of the ubiquitin cascade.

The method may be used for detecting complex formation of a ubiquitin moiety to a target moiety by covalent bond formation or molecular binding reaction. Accordingly, conjugation of the ubiquitin moiety to the target moiety may correspond to complex formation of a ubiquitin moiety to a target moiety by covalent bond formation or molecular binding reaction.

Alternatively, the method is used for detecting the removal of a ubiquitin moiety from a target moiety by a DUB. For example, the method can be for detecting the removal of ubiquitin from a substrate protein or a component of the ubiquitin cascade.

'Detecting' suitably comprises measuring the amount of the labelled ubiquitin moiety located on the surface. Detecting can be quantitative, or it may be qualitative. In some preferred embodiments the method can be used for detecting the ability of a test agent to modulate conjugation of a ubiquitin moiety to the target moiety or removal of a ubiquitin moiety from the target moiety. In such cases the method comprises providing a test agent, e.g. during step c) above, and measuring the effects of the test agent on the rate or extent of ubiquitin moiety conjugation/removal, e.g. compared to a control.

For example, the method can be used to determine if a test agent is an antagonist of the conjugation of a ubiquitin moiety to the target moiety (e.g. an inhibitor of ubiquitination). Alternatively, the method can be used to determine if a test agent is an agonist of the conjugation of a ubiquitin moiety to the target moiety (e.g. a promoter of ubiquitination). Likewise the method can be used to determine if a test agent is an antagonist or agonist of deubiquitination.

The method can be used to determine if a test agent is an allosteric modulator of an enzyme involved in the ubiquitination/de-ubiquitination pathway.

The test agent can be any suitable agent, including, but not limited to, chemical

compositions, such as small molecules, peptides, proteins, nucleic acids, lipids,

carbohydrates or any other chemical or biological agent suitable in applied molecular recognition science.

The target moiety can suitably be any moiety which is susceptible to conjugation to a ubiquitin moiety, or which is to be tested for its ability to be conjugated to a ubiquitin moiety. For example, the method can be used to investigate the conjugation of a ubiquitin moiety to any target protein of interest.

In some embodiments the target moiety is a ubiquitin-activating enzyme (E1), a ubiquitin- conjugating enzyme (E2) or a ubiquitin ligase (E3).

In other embodiments the target moiety is a substrate or putative substrate of ubiquitination, i.e. a protein which is ubiquitinated, or thought to be ubiquitinated, in vivo. A plurality of target moieties can be investigated in the method, e.g. several substrates of ubiquitination, several different E2s or E3s, or a combination of one or more substrates and one or more components of the ubiquitination pathway in multiplexed assays.

Thus in embodiments of the present invention the surface (e.g. bead) can have one of the following bound to it:

an E1 enzyme,

an E2 enzyme,

an E3 enzyme, and/or

a protein that is susceptible to ubiquitination, or where its susceptibility to ubiquitination is to be determined. Depending on the target moiety, the appropriate component or components of the ubiquitin system necessary for conjugation/removal of the ubiquitin moiety to/from the targeted moiety will be provided in the medium. For example:

Where activation of an E1 is to be investigated, the E1 can be bound to the surface and the medium can be provided with a labelled ubiquitin moiety and ATP. Thus, in this embodiment the component or components of the ubiquitin system can be merely ATP. Where charging of an E2 is to be investigated, the E2 can be bound to the surface and the medium can be provided with a labelled ubiquitin moiety, ATP and E1.

Where transfer of a ubiquitin moiety to an E3 (and thereby 'loading' it) is to be investigated, the E3 can be bound to the surface and the medium can be provided with labelled ubiquitin, ATP, and an appropriate E1 and E2. This reaction is only possible for HECT and HECT-like E3 enzymes that accept ubiquitin on the active site cysteine before transferring it to the substrate. Therefore, this reaction can be monitored with UPS-CONA.

- Where ubiquitination of a substrate protein is to be investigated, the substrate protein can be bound to the surface, and the medium can be provided with a labelled ubiquitin moiety, ATP, and an appropriate E1 , E2 and, if needed, E3.

Where deubiquitination of a substrate protein is to be investigated, the substrate protein can be bound to the surface, modified with the labelled ubiquitin moiety, and then exposed to a specific DUB enzyme in solution form.

The medium can be, for example, a cellular lysate from a desired organism or cell type. Such a cell lysate will endogenously contain physiological concentrations of various ubiquitination components, which can be supplemented with added exogenous labelled ubiquitin moiety, or endogenously expressed labelled ubiquitin moiety. In some cases additional ATP may be added.

The above examples each relate to media where the full complement of ubiquitination cascade members and associated factors are provided to drive the conjugation of a ubiquitin moiety to the target moiety of interest. However, it will be apparent that there may be circumstances where it is desirable to avoid providing such a complex mixture of agents, e.g. to ensure that a test agent is indeed modulating the reaction of interest (i.e. E3 mediated transfer to the target moiety), and not, for example, modulating a member of the pathway further upstream. E.g. in the fourth example above, it may be difficult to determine whether a test agent is modulating the transfer of the ubiquitin moiety to the substrate, or is modulating one of the upstream loading, charging or transfer steps. In such cases it may be preferable to provide a medium that contains the immediate upstream ubiquitin system member in a form in which it is already activated, for example, E3 already loaded with a ubiquitin moiety.

Accordingly, the method may be a method for determining which step of the ubiquitination pathway a test agent modulates. For example, the method may allow the determination of whether a test agent modulates the activation of the ubiquitin moiety by a ubiquitin-activating enzyme (E1), the interaction of E1 with a ubiquitin-conjugating enzyme (E2), or the transfer of the ubiquitin moiety to a protein substrate by a ubiquitin ligase (E3). Once the step modulated by the test agent has been identified, the method may be a method for determining the specific target of the test agent. For example, where a test agent has been found to modulate the interaction of the ubiquitin moiety with E1 , the method may allow the specific E1 with which the test agent interacts to be identified.

Accordingly, complex binding, activation or competition reactions, on substrate or allosteric interaction sites may be identified using the method of the invention.

It will be apparent that ATP is required for various key steps in the ubiquitination pathway. Accordingly, ATP is typically always present in the medium to permit conjugation reactions to occur. The negative control for the ATP-dependent reactions is realised in the absence of ATP. The addition of ATP can also be used as a convenient trigger to begin a given reaction; this can be particularly useful where the timing of progress of a reaction is important.

In embodiments of the present invention adapted to investigate ubiquitination (e.g. those defined in step b), part i) of the general method set out above), a DUB can suitably be provided in the medium. This allows, for example, for the effects of a test agent on the de- ubiquitination process to also be investigated. It may also make the system more

representative of the dynamic nature of parallel ubiquitination and de-ubiquitination events which occur in vivo. Likewise, where de-ubiquitination is to be investigated (i.e. in step b), part ii) of the general method set out above), one or more components which promote ubiquitination can be provided.

Where beads are used in the present invention, they can be any suitable size or shape, and formed from any suitable material. Various methods of manufacturing beads for use in such assays are known in the art, and many can be obtained commercially. Typically, suitable beads must be sufficiently hydrophilic to be incubated in test buffer solution and to form a monolayer of beads in a well plate. Such beads are typically formed from polymer resins, e.g. PEG hybrid polystyrene resin, or PEG-based polyamide resins. For example, nickel nitrilotriacetic acid (Ni²⁺NTA) agarose micro-beads (Life Technologies) can be used, as exemplified extensively below. TentaGel resin (a PEG hybrid polystyrene resin) or PEGA beads (e.g. those available commercially from RAPP Polymere or Sigma Aldrich) are preferred in some cases, and have advantages in terms of uniformity compared with agarose beads.

Preferably the beads are substantially spherical. However, other shapes (such as spheroidal or discs or the like) could be used in certain cases. The sphericity of a particle can be defined as the ratio of the surface area of a sphere (with the same volume as the given particle) to the surface area of the particle.

where V_p is the volume of the particle and A_p is the surface area of the particle. The sphericity of a perfect sphere is 1 and, any particle which is not a perfect sphere has a sphericity less than 1. Preferably, in embodiments of the invention where sphericity is important, the particles of the present invention have a sphericity of 0.95 or higher, such as 0.97 or higher, 0.98 or higher, or 0.99 or higher. The particles for use in the present invention preferably have a major dimension of from 1 to 1000 μηι, more preferably from 10 to 1000 μηι, yet more preferably from 20 to 250 μηι. By 'major dimension' is meant the largest dimension of a given particle, e.g. the diameter of a spherical particle, or the widest dimension of a plate-like particle. Where substantially spherical beads are used, the beads suitably have a diameter of from 10 to 1000 μηι, more preferably from 20 to 250 μηι. Beads having a diameter of approximately 60 μηι have been extensively exemplified below, but other sizes of beads would work perfectly well in various contexts. It is, however, preferred that the beads or other particles for use in the present invention have a major dimension of less than 250 μηι so that they can be easily manipulated and provide a reasonably miniaturised setup. Preferably the particles have a substantially uniform size distribution, e.g. wherein the population of particles has a coefficient of variance of less than 0.2, more preferably less than 0.1. The particle size of a spherical object can be unambiguously and quantitatively defined by its diameter. In some embodiments it is preferably that the beads are substantially uniform in size. In such cases it is generally preferred that the uniform, spherical polymeric beads exhibit a particle size distribution having a coefficient of variance of less than 0.2, more preferably less than 0.1.

However, for particles which are irregular in shape and non-spherical, the diameter cannot be applied, and, as discussed above, particles that can be used in the present application could be irregular in shape, plate-like, polyhedral or suchlike. In such cases having a uniform size will still be useful in many embodiments. There are several ways of extending the above quantitative definition of size for spherical particles, so that a definition is obtained that also applies to non-spherical particles. Most definitions are based on replacing a given particle with an imaginary sphere that has one of the properties identical with the particle. In the present invention, for particles which are non-spherical the major dimension can suitably be used; this is particularly useful for flat particles of particles which cannot be approximated to spheroids. Alternatively for particles which are relatively spherical, a volume based particle size can be used, in which the diameter stated equals the diameter of the sphere that has same volume as a given particle. Methods for determining particle size are well- known in the art, e.g. sieve analysis or optical granulometry. Preferably the particle size (be it the major dimension or volume based particle diameter) of the particles has a coefficient of variance of less than 0.2, more preferably less than 0.1.

Uniform particles size distributions within a population of particles can either be achieved by controlled manufacturing processes, or by sorting of a heterogeneous mixture of particles to isolate a population of the desired size distribution.

The target moiety can be attached to the surface by any suitable means, e.g. any suitable chemical or biological means. For some applications it may be preferable that the attachment is reversible to allow the target moiety to be detached from the surface easily. In one example the target moiety is provided with a coupling means and the surface is provided with a corresponding coupling means. For example, the surface can be provided with a nickel or cobalt complex and the target moiety can be provided with a tag which binds to nickel complex, e.g. a polyhistidine tag, such as a 6xhis or 10xhis tag. The nickel or cobalt can be conveniently provided in association with a chelator such as iminodiacetic acid (Ni ²⁺-IDA) and nitrilotriacetic acid (Ni²⁺-NTA) for nickel and carboxylmethylaspartate (Co²⁺- CMA) for cobalt. Other coupling mechanisms are known in the art, such as the biotin/avidin system, other protein affinity tags such as chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST), or peptide tags such as calmodulin tag, FLAG tag, Myc tag, Softag, Xpress tag, SBP tag, etc. It is also possible that the target moiety could be bound covalently to the surface. The person skilled in the art can select any suitable attachment technique from the many that are well-known in the art.

It will be apparent that, in some cases, it is desirable that the target moiety is modified, e.g. by recombinant or chemical techniques. For example, the target moiety can be a fusion protein in which a tag or other coupling means has been added. The target moiety could be modified by recombinant techniques to add a fluorescent protein marker, e.g. GFP or YFP. Alternatively, the target moiety could be chemically modified, e.g. to modify one or more amino acid side chains to facilitate coupling of the moiety to the surface, or to add a marker (e.g. a fluorescent chemical moiety). Such modifications should be carried out with care to avoid modifying the site of ubiquitination or sites of interactions with enzymes involved in the reaction.

A ubiquitin moiety can be labelled with any suitable label which permits detection of the ubiquitin moiety to the surface of the surface. Optical labels are preferred but radioactive or other markers may have utility in some embodiments. Fluorescent labels (fluorophores) are preferred as they are well suited to optical detection and can readily be attached to the ubiquitin moiety using well-known techniques.

It should be noted that, in some cases, it may be desirable to assess conjugation or removal of more than one type of ubiquitin moiety (e.g. ubiquitin, mutant versions of ubiquitin defective for formation of particular chain linkages, Nedd8, SUMO, etc.) in a multiplexed assay. In this case each type of ubiquitin moiety can be labelled with a label which allows each type of ubiquitin moiety to be distinguished from the other types. For example, a plurality of fluorophores with different emission wavelengths can be used to allow several ubiquitin moieties to be distinguished. The target moiety can also be labelled with any suitable label that permits detection, and this may be highly desirable in some embodiments, as described in more detail below. A highly suitable fluorescent marker for use in the present invention is fluorescein isothiocyanate (FITC), and other suitable fluorophores are set out below:

• Xanthene derivatives: e.g. fluorescein, rhodamine, Oregon green, eosin, and Texas red Cyanine derivatives: e.g. cyanine, indocarbocyanine, oxacarbocyanine,

thiacarbocyanine, and merocyanine.

Naphthalene derivatives (dansyl and prodan derivatives).

Coumarin derivatives.

Oxadiazole derivatives: e.g. pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole

• Anthracene derivatives: e.g. anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange.

Pyrene derivatives: e.g. cascade blue etc.

Oxazine derivatives: e.g. Nile red, Nile blue, cresyl violet, oxazine 170 etc.

• Acridine derivatives: e.g. proflavin, acridine orange, acridine yellow etc.

• Arylmethine derivatives: e.g. auramine, crystal violet, malachite green.

· Tetrapyrrole derivatives: e.g. porphin, phthalocyanine, bilirubin.

In some embodiments of the present invention, several fluorophores can be used in combination to label various different moieties. Detection of the conjugation or removal of the labelled ubiquitin moiety can be carried out by any suitable means adapted to detect the presence and location of the labelled ubiquitin moiety. Conjugation of the ubiquitin moiety to the target moiety will result in an accumulation of the label at the surface, and the method suitably detects the accumulation of said marker and thereby measures the amount of conjugation which has occurred. Likewise, when ubiquitin is removed from a target moiety linked, there will be a decrease in the amount of label at the surface.

Suitably the detection is carried out by an optical detection method, preferably by fluorescence detection. In preferred embodiments detection is carried out by confocal imaging, e.g. confocal microscopy. Confocal microscopy conveniently allows the user to view the location of fluorescent markers, and to make qualitative assessments and quantitative calculations. In the case of a surface where fluorescently labelled ubiquitin moieties have been conjugated to target moieties located on the surface, confocal microscopy will allow a layer of fluorescence to be detected at or near the surface. In the case of beads or other particles where fluorescently labelled ubiquitin moieties have been conjugated to target moieties located on the bead/particle surface, confocal microscopy will allow a 'halo' of fluorescence to be detected around the perimeter of a section though the bead/particle. The brightness of this layer or halo, detected as fluorescence emission intensity, will be determined by the amount of labelled ubiquitin moiety conjugated to the target moiety. The detection equipment is preferably a confocal fluorescence imaging system. An exemplary system is the Opera® system from Perkin-Elmer, but other systems are available commercially. The Opera® system is a high-content confocal microscope, which provides advantages in terms of speed, accuracy, and data evaluation. Preferably the detection equipment is configured to detect within a plane substantially parallel to the base of a vessel upon which beads are provided, and at a height above the base that falls within the diameter of beads from the base. Thus the detection equipment is focussed on a plane which provides a section through the beads. More preferably the plane is adapted to be approximately half of the diameter of the beads above the base, such that the detection is conducted in a section through the centre of the beads (i.e. the equatorial plane). For example, for beads having a diameter of 60 μηι, the detection ideally occurs at a height of about 30 μηι above the base, thus through the centre of the beads. In practice, it can be advantageous to use a plane which is slightly above or slightly below the equatorial plane (e.g. suitably from 1 % to 10% of the diameter above or below), which allows an image of the bead to be obtained which avoids that rings which appear attached to each other in the confocal image; this is useful because it allows better quantification of ring intensities when there is no overlap. Accordingly, in the above example, the detection may suitably occur at, for example, a height of from 24 to 29 μηι above the base. It will be apparent that this preferred aspect also applies to methods where particles other than beads are used.

In the case where the surface is a wall of a reaction vessel, then detection equipment can be configured to detect fluorescence at a suitable point. For example, this may be detection of fluorescence at or near the base of the vessel (which is typically substantially horizontal and parallel to the field of view of the detection equipment), or at or near the wall of the reaction vessel (which is typically vertical or sloped, and therefore oblique or normal to the field of view of the detection equipment).

The method can comprise measuring the progress of conjugation or removal of the labelled ubiquitin moiety over a period of time, e.g. by performing measurements at a plurality of pre- determined time points as the conjugation/removal process occurs or by measuring continuously. Such time-resolved monitoring of ubiquitination reactions can provide valuable information on reaction kinetics. Suitably the reactions occur in a suitable vessel. For example, the vessel can be a well in a multi-well plate, e.g. a micro-well plate, such as a 96, 384 or 1536 well microplate. Suitably the method is a high throughput method. For example, robotics can be used to automate the various steps of the method via technology well known in the art.

Suitably the method comprises monitoring multiple reactions in a single vessel. For example, a user can monitor conjugation of a ubiquitin moiety to an E2 and subsequently to a substrate protein. This can be achieved for example, by using two or more fluorescent labels with different emission wavelengths, e.g. a labelled ubiquitin moiety, a differently labelled E2 and a differently labelled substrate.

In some embodiments the method thus comprises using multiple fluorescent labels which emit light at different wavelengths, and are therefore distinguishable from each other. For example, the type of modification can be determined (such as mono- versus poly- ubiquitination), distinguishing between for example ubiquitination and neddylation, formation of polyubiquitin chains of given topology (K48, K63, etc.) or investigation of branched or mixed chains (ubiquitin, SUMO, Nedd8, etc.) by using differentially labelled lysine mutants or different modifiers.

The method may also comprise providing a plurality of surfaces. In embodiments comprising one or more particles, the plurality of surfaces may correspond to the surfaces of a plurality of particles. Preferably, the plurality of surfaces are provided in the same reaction vessel.

The method can also comprise providing two or more populations of surfaces (e.g. beads), which can be mixed or otherwise provided together (e.g. as an array on a surface), with each population having a different target moiety. For example, a first population can have a first target moiety bound on the surface of a first set of beads, the first target moiety being labelled with a first fluorescent label, and a second population can have a second target moiety bound on the surface of a second set of beads, the second target moiety being labelled with a second fluorescent label. A ubiquitin moiety having a third fluorescent label can then be used to detect preferential binding of the ubiquitin moiety to the first or second moieties. Such a method can be carried out where the first, second and third labels can be distinguished from one another, e.g. because they emit light at different wavelengths. An exemplary medium for use in the present invention is a buffer which comprises 50 mM Tris-HCI [pH 7.5], 5 mM MgC and 5 mM ATP in water. To this medium various

components of the ubiquitination system can be added. Other suitable media can, of course, be used.

In one embodiment of the present invention, there is provided a method for screening for modulators (inhibitors or promoters) of ubiquitination of a substrate protein, the method comprising providing said substrate protein linked to a surface, and observing for effects of a test agent upon ubiquitination. For example, a substrate protein of interest (e.g. p53) can be linked to beads, which are provided in a plurality of suitable reaction vessels (e.g. wells of a multi-well plate, such as a 96 well plate). The plurality of reaction vessels contain a suitable medium conducive to ubiquitination of the substrate protein, e.g. a cell lysate or synthetic medium containing E1s, E2s, E3s and ATP, and labelled ubiquitin moiety. Each reaction vessel is exposed to a test agent, typically from a library of compounds of interest.

Alternatively, each reaction vessels could be exposed to a sub-library containing a plurality of test agents, which is a sub-set of a complete library. Where an effect of interest is observed (e.g. an increase or decrease in ubiquitination levels or conjugation rates compared to a control), the test agent or sub-library is selected for further investigation. Where a sub-library is used, further screens may be required to determine which specific agent from the sub-library was effective. Of course, several replicates of reactions involving a given test substance or sub-library may be carried out to improve data quality; for example, in a 96-well plate.

In another embodiment of the present invention, there is provided a method for investigating details of the mechanism of ubiquitination of a substrate protein. In such an embodiment several reaction vessels can be used, the reaction vessels hosting reactions which correspond to different steps in the ubiquitination pathway which are to be investigated. This can be useful, for example, to identify where in the ubiquitination pathway a given modulator exerts its effect, e.g. whether it affects activities associated with E1 , E2, or E3. For example, a first reaction vessel could have E1 linked to beads, a second reaction vessel could have E2 linked to beads, a third reaction vessel could have E3 linked to beads (if the class of E3 is suitable), and a fourth reaction vessel could have a substrate protein linked to beads. The relevant ubiquitination components are provided in the medium to permit ubiquitination of the target moiety, with the ubiquitin being appropriately labelled. The conjugation of ubiquitin to E1 , E2, E3 or the substrate protein can therefore be detected and measured, and the effects of a given test agent on each individual part of the pathway assessed. This allows one or more test agents to be assayed separately against various parts of the ubiquitination pathway, thereby allowing the test agent's action to be better understood. Furthermore, it allows for several or all key steps in the ubiquitination pathway to be individually assayed on a single multi-well plate, which is ideal for high throughput investigations. For example, each different step of the ubiquitination pathway being examined could be set out in a different column of a multi-well plate, e.g. a first column for activation of E1 , a second for charging of E2, etc.

In another embodiment of the invention, the kinetics of various enzymes of the ubiquitination pathway can be studied. For example, a method can investigate the kinetics of charging of several different E2s. Each reaction vessel can contain a single E2 (or several E2s if several distinguishable labels are used) linked to a surface and the rate of charging of the different E2s with one or more labelled ubiquitin moieties can be determined.

In another embodiment of the present invention, a plurality of target moieties (e.g. three) are each labelled with a different and distinguishable fluorophore and a ubiquitin moiety is labelled with yet another different and distinguishable fluorophore. For example, E1 , E2 (potentially also E3) and a ubiquitin moiety can each be labelled with a different fluorophore, each fluorophore having an emission wavelength which is distinguishable from the others. The differentially labelled target moieties (e.g. E1 , E2 and E3, if suitable) can each be linked to suitable surfaces (e.g. beads), and the three populations of beads can be placed in a single reaction vessel. Where ubiquitin is conjugated to one of the three target moieties, this can be detected as a characteristic change in ratio of the fluorescence emission of the label used on E1 , E2 or if feasible E3, and the fluorescence emission of the label used on ubiquitin. For example, if E1 is tagged with a blue (B), E2 with a green (G), E3, with a yellow (Y) and ubiquitin with a red (R) emitting dye, the ratio changes of R/B, R/G or R/Y will provide a precise method of detection of relative ubiquitinations. Through this system, the detailed kinetics and maybe even the mechanism of various ubiquitination pathways can be interrogated. In another variant, several different ubiquitin moieties (e.g. ubiquitin, ubiquitin linkage mutants, Nedd8, SUMO, etc.) can be labelled with different and distinguishable fluorophores. Where polyubiquitination occurs, information about the composition of the heterogeneous chains can be determined by the mixed colours produced. This enables, for example, the preferred chain compositions generated by a various E2/E3 combinations to be compared.

It will be apparent that a great many permutations of different coloured labels are possible, and the above are just a small selection of the various possibilities contemplated by the present invention. Likewise an enormous variety of experimental systems can be designed depending on the various components linked to beads and provided in solution, and the person skilled in the art would be able to design experiments to probe many aspects of the UPS system based on the various examples described.

In a preferred embodiment of the present invention, the method is a screen for identifying one or more lead compounds for treatment of a disease. Many diseases are believed to be at least partly influenced by the UPS system. For example, the method can be for identifying lead compounds for the treatment of at least one of the following conditions and use in the fields of medicine and diagnostics:

- Cancer;

- Microbial infection: for example, bacterial or viral infection;

- Immune disorders: for example, autoimmunity and inflammation;

- Bone disorders;

- Cardiovascular disease;

- Regenerative medicine: for example, stem cell proliferation and renewal; and

- Neurological disorders: for example, Alzheimer's and Parkinson's diseases.

The method may screen a plurality of test agents to identify one or more lead compounds.

According to a second aspect of the present invention there is provided an assay for investigating molecular interactions and enzyme activities related to the UPS, the assay comprising:

- at least one surface having a target moiety bound to at least a portion thereof; and - at least one medium comprising a labelled ubiquitin moiety and a component or

components of the ubiquitination reaction suitable to conjugate the labelled ubiquitin moiety to the target moiety; and/or

- at least one medium comprising a deubiquitinating enzyme (DUB) suitable to remove a labelled ubiquitin moiety provided on the target moiety.

Details of suitable surfaces (e.g. beads), ubiquitin moieties, target moieties, components of the ubiquitination cascade, media and the like suitable for use in the assay have been described in detail above with respect to the first aspect of the invention. It should be understood that features described in respect of the first aspect of the invention can be applied to the other aspects of the invention. Preferably the surface is a surface of a particle, e.g. a bead.

Suitably the target moiety is a component of the UPS, e.g. an E1 , an E2 or an E3, or the target moiety can be a substrate (e.g. a protein) which is a known or putative target of ubiquitination in vivo.

Preferably the medium comprises components of the UPS and other ingredients sufficient to permit conjugation of a ubiquitin moiety to the target moiety. Alternatively, or additionally, the medium may comprise a DUB. Suitably the assay comprises at least one reaction vessel to provide or receive the at least one surface (e.g. receive a bead) and the medium. In a preferred embodiment the assay comprises a multi-well plate defining a plurality of reaction vessels.

The reaction vessel(s) suitably receives the beads and media as set out in the various exemplary embodiments mentioned above.

Preferably the assay comprises detection equipment to detect conjugation/removal of the labelled ubiquitin moiety to/from the target moiety by detecting the labelled ubiquitin moiety located at the surface of the beads. The detection equipment preferably comprises a confocal fluorescence imaging apparatus.

It is therefore preferred that the reaction vessel is adapted for use with confocal fluorescence imaging. For example, the reaction vessel should have a flat base for a layer of beads to sit evenly upon, and should preferably have a transparent bottom such that the base can be viewed clearly by the imaging system. A well of a multi-well plate is an example of a suitable reaction vessel.

In a preferred embodiment the assay is configured for high throughput screening. For example, it can be provided with robotic apparatus to automate one or more steps of the assay method. Thus, in a preferred embodiment the assay comprises a robotic handling apparatus. Suitable robotic systems are well-known in the art.

In a further aspect there is provided an assay kit for investigating molecular interactions and enzyme activities related to the UPS, the assay comprising:

- at least one surface having a target moiety bound to at least a portion thereof or adapted for convenient binding of a target moiety bound to at least a portion thereof; and

- at least one medium comprising a labelled ubiquitin moiety; and - at least one medium comprising a component or components of the ubiquitination system suitable to conjugate the labelled ubiquitin moiety to the target moiety; and/or

- at least one medium comprising a deubiquitinating enzyme (DUB) suitable to remove a labelled ubiquitin moiety provided on the target moiety.

Details of suitable surfaces (e.g. beads), ubiquitin moieties, target moieties, components of the ubiquitination cascade, media and the like suitable for use in the assay kit have been described in detail above with respect to the first aspect of the invention. Preferably the surface is a surface of a particle, e.g. a bead.

Such an assay kit can be provided to allow a user to conveniently carry out an assay method as described above. The labelled ubiquitin moiety and components of the ubiquitination cascade/deubiquitinating enzyme can be provided in the same portion of medium or in separate portions of medium. In some cases keeping the labelled ubiquitin moiety and the components of the ubiquitination cascade/deubiquitinating enzyme separate until the assay method is carried out may be advantageous.

The kit can suitably comprise a complex medium such (e.g., cell lysate) that lacks a particular enzyme activity, for example due to a genetic mutation in a patient.

In a still further aspect of the invention there is provided a method for investigating molecular interactions and enzyme activities related to the ubiquitin-proteasome system (UPS), the method comprising:

a) providing at least one surface with a ubiquitin moiety bound to at least a portion thereof; b) i) exposing said surface to a medium comprising a labelled target moiety and a

component or components of the ubiquitination system suitable to conjugate the ubiquitin moiety to a labelled target moiety; and/or

ii) wherein said ubiquitin moiety is conjugated to a labelled target moiety exposing said at least one surface to a medium comprising a deubiquitinating enzyme (DUB) suitable to remove the ubiquitin moiety from a labelled target moiety;

c) providing conditions permissive for the conjugation/removal of the ubiquitin moiety

to/from the labelled target moiety; and

d) detecting conjugation and/or removal of the labelled target moiety by detecting labelled target moiety located at or near the surface. The present method thus allows interrogation of ubiquitination and/or de-ubiquitination pathways (as set out in step b), parts i) and ii)).

Details of suitable surfaces (e.g. beads), ubiquitin moieties, target moieties, components of the ubiquitination cascade, media and the like suitable for use in the method of the present aspect have been described in detail above with respect to the first aspect of the invention. It should be understood that features described in respect of the first aspect of the invention can be applied to the present aspect of the invention. Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the accompanying drawings.

Brief Description of the Figures

Figure 1 : The ubiquitination cascade. Ubiquitin is first activated as a thioester linkage with an E1 , or ubiquitin-activating enzyme, in the presence of ATP which provides an energy source. In the second step, E1 interacts with an E2, or ubiquitin-conjugating enzyme and ubiquitin is transferred to the E2. Finally, an E3 ligase facilitates the ubiquitin transfer to a substrate protein. Multiple rounds of ubiquitination result in formation of a poly-ubiquitin chain. Various chain topologies due to the use of different lysine residues on the surface of ubiquitin influence the substrate protein function and fate. Deubiquitinating enzymes (DUBs) can remove ubiquitin from the substrate.

Figure 2: Excitation and emission spectra for fluorophores tested in UPS-CONA, overlayed with excitation and detection settings used on the Opera® (Perkin-Elmer).

Top charts: Excitation spectra of eCFP, eGFP, 5-TAMRA and Cy5 were overlayed with excitation lasers (445 nm, 488 nm, 561 nm and 640 nm, respectively). Bottom charts:

Emission spectra of eCFP, eGFP, 5-TAMRA and Cy5 were overlayed with detection windows (Camera 3: 475/34 nm, Camera 1 : 520/35 nm, Camera 2: 585/40 nm, Camera 2: 690/70 nm, respectively). The charts were generated using the Filter Selection Tool (Perkin Elmer) and Excel.

Figure 3: Concept of the UPS-CONA assay. Confocal Nanoscanning (CONA) is an on- bead screening technique developed in the lab of the present inventors for identification of small molecular ligand-protein interactions. CONA is based on confocal imaging of fluorescently tagged proteins bound to functionalised micro-beads. In a CONA experiment the confocal focus of a microscope adjusted to detect molecular interactions with single molecule resolution is positioned slightly below the equatorial plane of the bead. The spatial fluorescence intensity profile of a < 5 μηι thick layer of protein surrounding the bead in the image plane is collected through a cross-section that gives an intensity-enhanced exterior ring when binding occurs. A target protein is immobilised on micro-beads and incubated with appropriate ubiquitination reaction components, including fluorescently labelled ubiquitin. In the presence of ATP as energy source, ubiquitin is conjugated onto the on-bead substrate and the bead surface becomes fluorescent. On a confocal microscope, the ubiquitin conjugation can be visualised as fluorescent rings appearing around the beads. The bead profiles give information about the fluorescence intensity, corresponding to the extent of ubiquitin conjugation. The illustration on the bottom shows possible arrangements of the substrate on bead and components in solution.

Figure 4: Ubiquitin activation with E1 observed using UPS-CONA. E1 (Ubel) was immobilised on beads and incubated with FITC-Ubiquitin with or without ATP. The images were taken on the Opera® (Perkin-Elmer) after 6 h of incubation at x20 magnification and the ring intensities were analysed with ImageJ.

Figure 5: UPS-CONA is suitable for detection of ubiquitin- and Nedd8-conjugating enzyme activity. Beads with bound E2s (Ube2C, Ube2D2, Ube2L3, Ube2M, Ube2R1 , Ube2U) were incubated with FITC-Ubiquitin or FITC-Nedd8 and Ube1 (E1 for ubiquitin) or NAE (E1 for Nedd8) with or without ATP. The images were taken on the Opera® after 2 to 6 h of incubation. See Materials and Methods for conditions. The fluorescent ring formation was detected only when the active enzyme was used (i.e., Ube2U but not the inactive mutant Ube2U-C89A), in the presence of ATP as energy source (dotted line: no ATP, continuous line: with ATP).

Figure 6: Ubiquitin transfer to a HECT E3 ligase can be detected with UPS-CONA. E6AP HECT ubiquitin E3 ligase was immobilised on beads and incubated with FITC- ubiquitin, E1 (Ube1), E2 (Ube2D2), with or without ATP. Images were acquired after 6 h of incubation on the Opera® HCS instrument. See Materials and Methods for conditions.

Figure 7: Schematic illustration of experimental setup for monitoring full

ubiquitination reaction of an on-bead substrate protein, and UPS-CONA-based detection of substrate ubiquitination. p53 was immobilised on bead and incubated with components of the ubiquitination reaction: FITC-Ubiquitin, Ube1 (E1), E2D2 (E2), Mdm2 (E3) with or without ATP. The images were taken on the Opera® HCS instrument after 6 h of incubation. See Materials and Methods for conditions.

Figure 8: Detection of ubiquitin transfer from on-bead E1 to Cdc34 in solution. Ube1 was immobilised on bead and incubated with FITC-Ubiquitin, with or without ATP, in the absence or presence of the E2 enzyme Cdc34 (Ube2R1). The images were taken on the Opera® HCS instrument after 6 h of incubation. See Materials and Methods for conditions. Figure 9: PYR-41 dose-dependent inhibition of Ube1 activity detected with UPS-CONA. Cdc34 was immobilised on bead and incubated with FITC-ubiquitin, Ube1 and increasing concentrations of the E1 inhibitor PYR-41 as indicated. The images were taken on the Opera® HCS instrument after 6 h of incubation. See Materials and Methods for conditions. Figure 10: MLN4924 dose-dependent inhibition of NAE activity detected using UPS- CONA. On-bead Ube2M was incubated with FITC-Nedd8, NAE, ATP and increasing concentrations of MLN4924 as indicated. DMSO-only samples were used as positive (without ATP) and negative (with ATP) controls. The ring intensities were compared and represented as bead profiles. The images were taken on the Opera® HCS instrument after 6 h of incubation. See Materials and Methods for conditions.

Figure 11 : Nutlin-3 inhibits p53 ubiquitination in a concentration-dependent way. p53 was immobilised on bead and incubated with FITC-ubiquitin, Ube1 (E1), Ube2D2 (E2) and Mdm2 (E3), with or without ATP and DMSO or 100 μΜ of nutlin-3. The images were taken on the Opera® HCS instrument after 8 h of incubation. See Materials and Methods for conditions.

Figure 12: Ubiquitin-charging reaction in vitro can be monitored in real-time.

On-bead E2 (Cdc34) was incubated with the ubiquitination reaction including a fluorescent FITC-ubiquitin and observed on the Opera®. Maximal fluorescence intensity was measured in a randomly chosen field over 8 h. An exemplary view on the beads was represented below for chosen time-points. The bead profiles, representing the fluorescence intensity along a bead diagonal, were compared from a representative bead at 0, 1 and 5 hours. Figure 13: Double colour detection of ubiquitin charging onto Ube1. AlexaFluor 633- labelled Ube1 was attached to the beads and incubated with FITC-ubiquitin with or without ATP. Three different channels on the Opera® HCS instrument were used to detect beads (LED, brightfield), on-bead Ube1 (633 nm, red) and ubiquitin (488 nm, green). In the well without ATP, only red rings were detected (Ube1) while in the well with added ATP, both colours red (Ube1) and green (conjugated FITC-ubiquitin) were detected.

Figure 14: Double colour detection of ubiquitin charging onto Cdc34. GFP (488) - Cdc34 fusion protein was attached to the beads and incubated with Ube1 and fluorescently labelled TMR ubiquitin, in the presence or absence of ATP. The 488 nm channel (green) was used to detect GFP-Cdc34 and the 561 nm channel (pink) for detecting TMR-ubiquitin. An arbitrary cut-off of 500 AU for fluorescence intensity was applied to eliminate background fluorescence. In the well without ATP, only green rings were detected (Cdc34) while in the well with added ATP, both colours green (Cdc34) and pink (conjugated TMR-ubiquitin) were detected.

Figure 15: Detection of two distinct bead populations in one well. 6xhis-tagged Ube1 labelled with AlexaFluor 633 (red) and Cdc34 labelled with AlexaFluor 488 (green) were incubated separately on nickel-NTA agarose beads, extensively washed and combined in one well. Pictures were acquired on the Opera® HCS instrument using a set of lasers and filters as described in the Methods. Arrows point to the green or red rings, as indicated. Figure 16: Example of bead analysis using the BeadEval software (Evotec).

Ni2+NTA agarose beads were incubated with 6xHis-tagged eGFP and imaged on confocal Opera® HCS instrument. After scanning, the images were analysed with BeadEval software (Evotec), which generates an output picture with encircled recognised beads (left picture). The chart (right) represents the ring intensity distribution of beads within a well.

Figure 17: Illustration of an automatic, high-throughput screening setup using UPS- CONA. The UPS-CONA screening process will be executed in four steps: 1) Preparation of solutions, beads (and compounds for library screening) and mixing in a microplate, using an automated liquid handling robot 2) Incubation of the ubiquitination reaction for a required time 3) Detection on a high-throughput fluorescent confocal microscope such as the Perkin Elmer Opera® HCS instrument 4) Data analysis and potential hit identification. Potential hits would be analysed further in secondary on-bead or in solution assays.

Figure 18: Setup and detection of Cy5-ubiquitin conjugation onto the on-bead eGFP- Ube2C. eGFP-labelled Ube2C is immobilized on bead, while Cy5-ubiquitin is kept in solution together with the E1 enzyme. In the reaction buffer containing ATP, Cy5-ubiquitin is conjugated onto on-bead Ube2C. Both eGFP and Cy5 can be detected as fluorescent rings in the confocal plane of a fluorescence microscope (bottom left images). The ring intensity is proportional to the amount of bound fluorescent protein. The ratio between the on-bead fluorescent substrate protein and the conjugated ubiquitin can be used to identify hits and eliminate potential false positives, which can be identified as affecting intensity of both the on-bead substrate and ubiquitin signal.

Figure 19: Hit1 and Hit2 decrease the signal detected from Cy5-ubiquitin conjugation onto the on-bead eGFP-Ube2C. A: Representative images taken on the Opera reader from UPS-CONA detection of Cy5-ubiquitin conjugation onto on-bead Ube2C in the presence of Hit1 and Hit2. On-bead eGFP-Ube2C or unlabelled but His-tag

conjugatedUbe2C were incubated with 20 μΜ of Hit1 or Hit2 in a ubiquitination reaction containing Cy5-ubiquitin. The ubiquitination mix without ATP was used as negative control. The DMSO concentration was adjusted accordingly. Images from the Cy5 channel were acquired on the Opera (PE) and processed through a plate montage Acapella script. B: Ring intensity analysis. Opera images were analysed using ImageJ. Ring Cy5 fluorescence corresponding to the conjugated ubiquitin are represented as bead profiles for intensity comparison.

Figure 20: Hit1 and Hit2 affect Cy5 signal from ubiquitin charged onto on-bead E2s and E1. UPS-CONA was performed with on-bead Ube2L3, Ube2R1 and Ube1 with 20 μΜ Hit1 or Hit2 as indicated. Samples which did not contain ATP were used as control. A: Opera images were generated with the Acapella plate montage script. B: Bead profile analysis was performed using ImageJ and Excel.

Figure 21 : Analysis of the effects of hit analogues on eGFP and on Cy5-ubiquitin conjugation onto on-bead eGFP-Ube2C. 32 analogues affecting Cy5-ubiquitin signal and the initial hits were incubated with eGFP on bead or in the ubiquitination reaction with eGFP- Ube2C on bead and Cy5-ubiquitin in solution. Images were acquired on PS04 and data was analysed using ImageJ and Excel. Represented are the controls and the 9 compounds which least affected the eGFP signal, ranged by their effect on the Cy5-ubiquitin signal and the effects on eGFP, eGFP-Ube2C and Cy5-ubiquitin are shown.

Figure 22: On-bead CHIP exhibits autoubiquitination activity in UPS-CONA. NiNTA agarose beads were incubated with 6xHis-CHIP or buffer only and placed in wells of a 384- well plate together with components of the ubiquitination reaction as indicated.

Concentrations were used as in a standard UPS-CONA assay. Plate was visualised on the Opera instrument (Perkin-Elmer) and the images were analysed using an Opera Acapella script, ImageJ and Excel. The chart represents intensity profiles of a cross-section of a representative bead in each well.

Figure 23: Determination of the optimal substrate amount for UPS-CONA of CHIP and UbcH5a. 0, 5, 10, 15 and 20 pmoles per well of 6xHis-CHIP or 6xHis-UbcH5a were incubated with NiNTA agarose beads. Beads were then washed and placed in standard ubiquitination reaction including Cy5-Ub, Ube1 , UbcH5a (for CHIP only), in the presence or absence of ATP. Bead images were acquired with Opera (PE) and analysed using Acapella software, ImageJ and Excel.

Figure 24: Determination of the optimal E1 concentration for UPS-CONA of CHIP.

NiNTA beads were incubated with 6xHis-CHIP, washed, distributed in a microplate and incubated in a ubiquitination reaction with varying concentrations of Ubel Images were acquired on the Opera (PE) and analysed using Acapella, ImageJ and Excel.

Figure 25: Determination of the optimal E2 concentration for UPS-CONA of CHIP. NiNTA beads were incubated with 6xHis-CHIP, washed, distributed in a microplate and incubated in a ubiquitination reaction with varying concentrations of UbcH5a. Images were acquired on the Opera instrument (PE) and analysed using Acapella, ImageJ and Excel. Figure 26: Determination of the optimal ubiquitin concentration for UPS-CONA of CHIP. NiNTA beads were incubated with 6xHis-CHIP, washed, distributed in a microplate and incubated in a ubiquitination reaction with varying concentrations of Cy5-Ub. Images were acquired on the Opera (PE) and analysed using Acapella, ImageJ and Excel.

Figure 27: Parallel monitoring of mixed ubiquitination and neddylation reactions. On the left side of the image, one field of view from the well with on-bead Cdc34 is shown; on the righ side, one field of view from the well with on-bead Ubc12. The first row shows images acquired upon 488 nm excitation (blue), which allows detection of FITC from the Nedd8 label; the second row shows images acquired upon 640 nm excitation (pink), which allows detection of Cy5 from the ubiquitin label. The third row represents merged images with subtracted background. 640 nm ubiquitin rings are detected only on beads with Cdc34, while 488 nm Nedd8 rings are detected only on beads with Ubc12. Illustration on the right side explains the process where parallel monitoring of ubiquitination and neddylation cascade can be performed within a single well, if the substrate proteins can be differentiated by their distinguishable fluorophores (for example red and green). In this specific example, Cdc34 would be green, Ubc12 would be red; ubiquitination would be detected as formation of pink rings (from the Cy5 fluorophore on ubiquitin) and neddylation would be detected as formation of blue rings (from the FITC label on Nedd8).

Figure 28: Imaging of three differentially labelled E2s on-bead. Three different E2s were produced as different fluorescent protein conjugates, attached to agarose beads and beads were placed as approximate 1 : 1 :1 mix in a single well. Images represent the same field of view within the test well, taken with different excitation/emission settings, as described in the Materials and Methods section. The merged image lights up all beads detected in the field of view, artificially coloured according to the fluorescent protein code. Blue: mTurq2, green: eGFP, red: E2-Crimson. The illustration below shows a sketch of simultaneous detection of ubiquitin charging to three different E2s in a single well which has now proved to be feasible.

Specific Description of Embodiments of the Invention Introduction

Ubiquitin and ubiquitin-like modifiers

Ubiquitin

Ubiquitin is a small, 76 amino acid-long protein with a molecular weight of 8.5 kDa (Goldstein et al., 1975). The globular three-dimensional structure of ubiquitin (Vijay-Kumar et al., 1985) is highly conserved throughout the Eukaryotes. In the human genome, four genes encode for ubiquitin: UBB, UBC, UBA52 and RPS27A (Kimura and Tanaka, 2010). After expression, the gene products are processed and cleaved to produce mature forms of monomeric ubiquitin, characterised by a C-terminal diglycine sequence. Abundantly expressed in eukaryotic cells and in various tissues, ubiquitin proteins are conjugated to substrates or present in the form of free monoubiquitin or polyubiquitin chains, with the free pool used in regulatory functions dependent on cellular conditions. Ubiquitin-like proteins

Besides ubiquitin, 17 ubiquitin-like modifiers (UBLs) from nine different classes (NEDD8, SUMO, ISG15, FUB1 , FAT10, Atg8, Atg12, Urm1 , and UFM1) have been identified in human cells as being able to conjugate to substrates (reviewed in Schulman & Harper, 2009). UBLs exhibit variable sequences but similar structure, sharing a common "ubiquitin fold". However, different modifiers have their specific conjugation cascades and have diverse impacts on their substrates. The well-studied UBLs are SUMO and NEDD8. SUMO (Small Ubiquitin-like Modifier) proteins display 18% identity with ubiquitin and are around 100 amino acids in length. Four SUMO proteins have been identified in humans: SUMO-1 , SUMO-2, SUMO-3 and SUMO-4, with SUMO-2 and -3 being very similar in sequence and function. SUMO conjugation occurs on lysines within a specific consensus motif, ψΚχϋ/Ε, where ψ is a large hydrophobic amino acid and x any amino acid. The SUMO conjugation cascade involves a heterodimeric E1 , SAE1/Uba2 (SUMO-Activating Enzyme), a unique E2: Ube2l and a small number of identified E3 ligases, although it has been shown that E2 activity is sufficient if the sumoylation motif is present. SUMO also interacts non- covalently with other proteins via SIMs (SUMO-lnteracting Motifs). Mostly monomeric on in vivo conjugates, SUMO-2/3 are able to form polySUMO chains as well. DeSUMOylation reaction is driven by SENP proteinases. Sumoylation seems to be strongly interconnected with other post-translational modifications, including ubiquitin. SUMO conjugation regulates, among others, protein localisation and stability, cellular response to stress, transcription, DNA repair and cell cycle progression (Hay, 2005). Consequently, dysregulation of sumoylation is associated with diseases such as neurodegenerative disorders and cardiomyopathy (Jeon et al., 2011).

NEDD8 is a 81 amino acid polypeptide with 58% sequence identity to ubiquitin. The neddylation cascade comprises a heterodimeric E1 , APPBP1/Uba3 (Alzheimer-precursor protein-binding protein-1-ubiquitin-activating enzyme-3) or NAE (Nedd8-Activating Enzyme), two known E2s: Ube2M and Ube2F, and E3 ligases, such as Mdm2 or Dcn1 , accompanied by Rbx1/2 (Kurz et al., 2008), although more NEDD8 E3 ligases are likely to be discovered. A large spectrum of proteins have been identified as neddylation substrates, including cullins - components of the ubiquitin E3 ligase SCF complexes, ribosomal proteins, and other substrates involved in transcription, DNA repair and replication, and cell cycle regulation (Xirodimas, 2008). Interestingly, it has been reported that under cellular stress, the components of the ubiquitination cascade are used instead of the NEDD8-specific enzymes and NEDD8 is conjugated to ubiquitination substrates (Leidecker et al., 2012). NEDD8 has been reported to form chains in vitro, but biological function of these chains remains unknown. NEDD8 can be removed from substrates by the dennedylation enzymes, such as DEN/NEDP1 and the COP9 signalosome. Neddylation perturbations have implications in oncogenesis, and a NAE inhibitor is currently in clinical trials for various cancers (Nawrocki et al., 2012).

Functions for less well-known ubiquitin-like proteins have also been reported. ISG15

(Interferon-Stimulated Gene 15) is involved in interferon-induced pathways, in antiviral function (Ritchie and Zhang, 2004), similar to FAT10, which is also involved in proteasomal degradation (Hipp et al., 2005). UFM1 (Ubiquitin-Fold Modifier 1) regulates antioxidant pathways, while ATG7 (Autophagy-related protein 7) participates in autophagosome formation (Mizushima et al., 1998).

Prokaryotic ubiquitin homologs

Ubiquitin is highly conserved in eukaryotes, but prokaryotes also possess proteins with a ubiquitin-like fold, for example bacterial ThiS (thiamine biosynthesis protein S) and MoaD (molybdopterin-converting factor subunit 1) in E. coli, involved in sulphur transfer, which use a similar chemistry to ubiquitin conjugation. Nonetheless, MoaD and ThiS do not share sequence similarity with eukaryotic ubiquitin and are not involved in proteolysis. Interestingly, recent studies also revealed the existence of a ubiquitin-like polypeptide, which can be conjugated to other bacterial proteins as a marker for degradation. Pup, (prokaryotic ubiquitin-like protein), was shown to be specifically conjugated to lysines of proteasome substrates in pathogenic Mycobacterium tuberculosis (Pearce et al., 2008). Pupylation occurs though activity of proteasome accessory factor A (PafA) instead of a classic enzymatic cascade. This was the first demonstration of prokaryotic protein stability regulation via a ubiquitin-like modifier. Although Pup presents an unordered structure and has no sequence similarity to eukaryotic ubiquitin, it has been hypothesised that it might acquire a ubiquitin related beta-grasp fold upon conjugation to the substrate. Importantly, PafA was previously reported to cause lethal Mycobacterium tuberculosis infections in mice, which might suggest that the bacterial conjugation cascade is involved in pathogenesis and could be a potential clinically relevant target.

Until recently, clear differentiation could be made between the ubiquitin and ubiquitin-like proteins and their conjugation cascades in eukaryotes and relatively primitive ubiquitin-fold proteins in bacteria. However, the situation dramatically changed with the discovery of a eukaryotic ubiquitin homolog in human commensal bacterium Bacteroides fragilis (Patrick and Blakely, 2012; Patrick et al., 2011). With 76% identity to a gene from a migratory grasshopper virus, the B. fragilis ubiquitin was probably acquired via horizontal transfer of genes to the intestinal bacteria. Considerable questions remain as to whether and how this eukaryotic ubiquitin provides B. fragilis with a selective advantage and ability to interfere with the human UPS and consequently, with the host immune system, especially since the purified "bacterial" ubiquitin was able to block the human E1 in vitro. As one of the predominant bacteria in human gastrointestinal tract, B. fragilis and its aberrant eukaryotic ubiquitin might be implicated in inflammatory and autoimmune diseases.

Ubiquitin-activating enzymes: E1s

E1s initiate the ubiquitination cascade by activating ubiquitin to be further transferred to an E2 and a substrate. E1 activity may reside in a single, large-sized protein, such as ubiquitin E1s, or a heterodimer, in case of NEDD8 and SUMO E1 s. The conserved E1 structure contains an ATP-binding domain, responsible for initial interaction and adenylation of ubiquitin, a catalytic cysteine domain, where ubiquitin thioester is formed and a C-terminal ubiquitin-fold domain (UFD) responsible for interaction with E2s. E1 has two active sites, required for the two-step activation of ubiquitin. In the first, energy dependent step, ATP- Mg²⁺ and ubiquitin bind to the adenylation domain and the C-terminal glycine in ubiquitin is adenylated. The second step consists of a ubiquitin-AMP transfer onto the active site cysteine with release of AMP and formation of the ubiquitin thioester. Consequently, the activated E1 is simultaneously bound by two ubiquitin moieties, in form of thioester and adenylate.

Each ubiquitin-like modifier is activated by a specific E1 , although increasing evidence supports the existence of crosstalk between supposedly parallel pathways. Eight E1s have been identified so far in human cells. Ubiquitin is activated by Uba1 or Uba6, with the latter also activating FAT10 and specifically charges the E2 Ube2Z. The other most relevant E1s, include the heterodimers SAE1/Uba2 (SAE) and APPBP1/Uba3 (NAE) which activate SUMO and NEDD8, respectively, and Uba7 acts as an E1 for ISG15 (Interferon-stimulated Gene 15).

Ubiquitin-conjugating enzymes: E2s

The E2 enzymes interact with the ubiquitin E1 loaded enzyme and ubiquitin is transferred to an active site cysteine on the E2. The ubiquitin-charged E2 interacts in turn with an E3 and, depending on the E3 type, transfers ubiquitin to the E3 or directly contributes to ubiquitin attachment on a lysine residue of the substrate protein. However, E2s not only enable the ubiquitin transfer to the substrate, but also accomplish additional functions in determination of ubiquitin chain length and linkage type. The human genome encodes 38 different E2 enzymes for ubiquitin and UBLs.

Ubiquitin ligases: E3s

The eukaryotic E3s are classified into two major families: the RING (Really Interesting New Gene) (Deshaies and Joazeiro, 2009) together with the U-box ligases (Cyr et al., 2002), and HECT (Homologous to E6-AP Carboxyl Terminus) ligases (Metzger et al., 2012) (also RING/HECT or RING-in between-RING (RBR) E3s (Wenzel et al., 201 1a)). A third structurally distinct class of E3, termed the IpaH family, appears to be specific to bacterial pathogens (Rohde et al., 2007). In contrast to RING domain class of E3s, in which the RING domain provides a non-covalent binding surface for the E2, the HECT, RBR and IpaH E3s themselves contain an active site cysteine to which ubiquitin is transferred from an E2 before conjugation to a substrate. In the ubiquitination process, the E3 enzyme recruits a cognate E2 and ensures the substrate and linkage type specificity. Moreover, in many cases E3s prime allosteric activation of the E2, induce proper substrate positioning for ubiquitin transfer at a determined site and contribute to the polyubiquitin chain formation. Some E2s and E3s act in a ordered hierarchy, in which a priming E2 or E3 adds the first ubiquitin moiety, which is then extended by a second E2 or E3 (Koegl et al., 1999, Williamson et al., 2009). Other E3s depend on a prior SUMO modification of the substrate in order to recognize and ubiquitinate substrates ( Lallemand-Breitenbach et al., 2008).

Proteasome

The proteasome is a eukaryotic ATP-dependent protease, responsible for degradation of ubiquitin-tagged proteins (Hershko et al., 1984). This large, 2.5 MDa, multi-subunit complex contributes to regulation of most if not all cellular processes through selective protein degradation and aberrant proteasomal activity is associated with clinical outcomes (Tanaka, 2013). The 26S proteasome is composed of a 20S catalytic core and two 19S regulatory particles, forming the "lid" and the "base" of the proteasome. The regulatory particles recognise polyubiquitinated proteins and are able to cleave off the ubiquitin chains for recycling, prior to unfolding and translocating the substrate proteins into the catalytic core. The cylindrical catalytic subunit is composed of four heptameric rings, made of external alpha subunits and internal beta subunits. The beta subunits present specific peptidase activities, responsible for cleaving the substrate proteins. Deubiquitinating enzymes: DUBs

Ubiquitination is a dynamic and reversible process, as conjugated ubiquitin can be removed from the modified substrate by deubiquitinating enzymes, or DUBs. DUBs are able to cleave the isopeptide linkage at the C-terminus where ubiquitin is conjugated to the substrate or within a polyubiquitin chain, resulting in release of ubiquitin from the target protein, or in a shortened ubiquitin chain (Komander et al., 2009; Nijman et al., 2005). An estimated number of 80 human DUBs have been classified into five families with varying function and specificity (Amerik and Hochstrasser, 2004). Some DUBs are chain-linkage specific, while others target a specific target independently of the type of modification. The various DUB can protect specific ubiquitinated substrates from degradation by removing polyubiquitin chains before recognition by the proteasome. A proteasome-associated called Usp14 dictates the dynamics of substrate recognition by the proteasome by trimming poly-ubiquitin chains prior to full proteasome engagement of the substrate (Lee et al., 2010).

Biological functions of ubiquitination

Substrate proteins can be modified with ubiquitin on their exposed lysine residues or at their N-terminus, although non-canonical modifications on serines, threonines or N-termini have also been described (Bloom et al., 2003; Wang et al., 2007). A modification is called monoubiquitination when a unique ubiquitin entity is attached to the substrate. If multiple single ubiquitins are attached to several lysines, the substrate modification is termed multiubiquitination (or poly-monoubiquitination). Finally, polyubiquitination occurs when a substrate is modified with a chain of ubiquitin proteins linked through isopeptide bonds. Indeed, ubiquitin has seven lysine residues (K6, K1 1 , K27, K29, K33, K48, K63), which can be covalently linked to the C-terminal glycine of another ubiquitin (Peng et al., 2003), forming a polymer. Depending on the lysines that are used for attachment, different chain topologies are formed, which provide differential function to the substrate (Sadowski et al., 2012;

Welchman et al., 2005). Alternatively, ubiquitins may be linked via their C- to N-termini, forming a linear chain (Kirisako et al., 2006) and conferring yet another outcome for the target protein (Li and Ye, 2008). Different chain linkages are primarily determined by the E2 enzyme that is used in the reaction.

The table below shows the involvement of various types of ubiquitin modifications in cellular processes.

Monoubiquitination has been shown to regulate processes such as DNA repair, transcription and endocytosis and protein sorting (Haglund et al., 2003). The lysine 48-linked ubiquitin chains (K48) are the principal signal for protein degradation (Thrower et al., 2000), efficiently recognised by the proteasome, with at least four ubiquitins in the chain. Similarly, K1 1 chains and in some cases K63, K29 or K6 might also target a substrate for degradation (Jin et al., 2008). Proteasomal degradation can affect virtually all cellular functions; for instance, degradation of cell cycle-regulating proteins effects the control of cell cycle progression. K63 chains are implicated in protein endocytosis, DNA repair and signal transduction (Passmore and Barford, 2004), including NF-kB (Nuclear factor NF-kappa-B) activation. NF-kB activity is also regulated by linear ubiquitin chains (Tokunaga et al., 2009). The functions of the other chain types - K6, K27, K29 and K33 remain unclear. Some substrates are also modified with mixed chains comprised of more than one linkage type.

Ubiquitination in disease

With the rapidly increasing understanding of the UPS and its associated biological functions, a growing number of links between particular enzymatic activities and disease states have been established. Various diseases have been associated with aberrant functioning of the ubiquitination system, such as cancer, immune evasion of pathogens, neurodegenerative diseases and metabolic disorders including diabetes (Petroski, 2008). A few examples of mechanisms involved are given below. Identification of relevant clinical targets allows the subsequent search for small molecular modulators, which would help unravelling

mechanisms of diseases and might ultimately be developed into therapeutics.

Cancer p53 regulation

p53 is a transcription factor, involved in DNA damage response and apoptosis, and is a very common tumour suppressor, found to be mutated in 50% of cancers. Its stability and activity is regulated by an E3 ligase called Mdm2 (Murine double minute 2 protein homologue) through ubiquitination and neddylation. Moreover, p53 is implicated in cellular stress responses mediated by ribosomal proteins (Sundqvist et al., 2009). Additionally, p53 downregulation via pathological ubiquitination induced by human papillomavirus (HPV) interference has been linked to cervical cancer. Indeed, the HPV produces the E6 protein, which binds to a HECT E3 ligase for p53, E6AP (Human papillomavirus E6-associated protein), and promotes the recruitment of p53, its ubiquitination and degradation, leading to oncogenic progression (Wang et al., 2001). Therefore, p53 stabilisation and enhancement of activity, for example by inhibiting the ubiquitination activity of Mdm2 or enhancing the activity of the deubiquitinating enzyme HAUSP (Herpesvirus-associated ubiquitin-specific protease), are considered to be a viable approaches to target a large array of tumours.

EGFR signalling

EGF-induced signalling impacts on critical cellular processes, such as proliferation or apoptosis, which, if deregulated, have been shown to result in carcinogenesis. Upon cell stimulation with extracellular signalling molecules, such as epidermal growth factor (EGF) or transforming growth factor alpha (TGF-alpha), EGF receptors on the cell surface dimerise and activate downstream signalling. First, due to the intrinsic protein tyrosine kinase activity, the EGFRs autophosphorylate tyrosines in their cytoplasmic domains. Phosphotyrosines are recognised by the Cbl (Casitas B-lineage lymphoma proto-oncogene) family of E3 ligases, which in turn ubiquitinate the cytoplasmic regions of EGFRs. Next, specific kinases are recruited and transduce the signal to activate cellular pathways, which eventually lead to induction of genes regulating apoptosis, cell survival and proliferation. Ubiquitination regulates not only the initial EGFR phosphorylation, but, via modification of adaptor proteins such as EGFR substrate 15 (EPS15) or EPS15-interacting proteins (EPSINs), also contributes to the progression of the EGFR endocytosis and recycling.

Furthermore, the downstream signalling pathways initiated with EGFR are regulated by ubiquitination. In the case of the GTPases Ras, depending on the Ras isoform, the Ras protein ubiquitination can activate or inhibit the Ras cascade, controlling the oncogenic events in the cell. Moreover, the activity of a HECT E3 ligase ITCH, also regulates the cell response. ITCH ubiquitinates and contributes to degradation of proapoptotic proteins, such as p73 or tBid (truncated BH3 interacting domain), which leads to attenuation of the apoptosis and cell survival.

Degradation of cell cycle regulators

Cullin-RING ubiquitin ligases (CRLs) and the anaphase-promoting complex/cyclosome (APC/C) are RING ubiquitin ligases with well-established clinical relevance (Cardozo and Pagano, 2007) as essential modulators of cell cycle regulators. In particular, the SKP2 F-box protein, which contributes to degradation of CDN1 B (the cyclin-dependent kinase inhibitor p27) at the G1/S transition as part of a CRL complex called SCF, is considered as a potential oncology target because SKP2 is overexpressed in many human cancers

(Signoretti et al., 2002). Conversely, mutations downregulating the activity of another E3 ligase involved in regulation of the cell cycle and DNA damage response, the heterodimer BRCA1/BARD1 (Breast cancer type 1 susceptibility protein /BRCA1 -associated RING domain protein 1), have been linked with oncogenic outcome, and enhancement of this activity has been proposed as a therapeutic route against breast and ovarian cancers (Hashizume et al., 2001).

Immune evasion

Certain immune responses are also controlled by ubiquitination. Recognition of pathogens on the cell surface leads to activation of the NF-kB signalling cascade, resulting in ubiquitination by E3 ubiquitin ligase β-TrCP (beta-transducin repeat containing protein) and degradation of Ik-Ba (NFkappa-B inhibitor alpha), which eventually releases NF-kB into the nucleus to activate transcription of pro-inflammatory genes (Kanarek et al., 2010). Some viruses have been shown to hijack the host UPS to evade the immune system, including HIV. The viral protein Vif associates with the human Cullin-RING ubiquitin ligases to ubiquitinate and target for degradation an antiviral protein APOBEC3G (apolipoprotein B mRNA-editing, enzymecatalytic, polypeptide-like 3G) (Yu et al., 2003). Interestingly, the inhibition of cullin neddylation seems to restore the APOBEC3G activity and presents a novel therapeutic approach (Stanley et al., 2012). Similarly, it is likely that other pathogens, eukaryotic and bacterial, are able to overcome the host immune system by interfering with the UPS. Neurological disorders

Since the UPS ensures the intracellular quality control process and is responsible for removal of misfolded or damaged proteins also in the brain, it is not surprising that perturbation of the degradation processes might result in accumulation of ubiquitinated proteins toxic to the cells, which is a characteristic of the major neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases (AD, PD). Although the role of the UPS in neurodegenerative disorders are still under investigation, it has been suggested that initial perturbations of the UPS may lead to accumulation of aberrant proteins and/or, that the formation of protein aggregates resistant to proteolysis may eventually impair proteasome function (Upadhya and Hegde, 2007). Additionally, loss of function mutations in Parkin, an E3 ligase specifically involved in PD, has been indicated as a major factor in familial PD, suggesting that its malfunction could contribute to the development of the disease (Imai et al., 2000). Ubiquitin-proteasome system as drug target

The role of the UPS in disease states and its clinical relevance are now undeniable. The parallel between protein ubiquitination and phosphorylation has been drawn in terms of the possibilities for new clinical targets, drug discovery and eventually, novel therapies (Cohen and Tcherpakov, 2010). So far, the number of discovered chemical modulators of specific ubiquitination and deubiquitintion reactions has been limited, but increasing interest from basic research to the pharmaceutical industry raises hopes for exciting developments in the future.

The FDA approval of a proteasome inhibitor bortezomib (Velcade) in 2003 for treatment of multiple myeloma confirmed the clinical potential of the UPS. Although inhibition of proteasomal activity might seem unspecific in terms of outcome, as degradation of many proteins could be impaired in both tumorous and normal cells, bortezomib exhibited relatively high specificity in affecting cancer cells, which produce more aberrant and oncogenic proteins than normal cells. In 2012, another proteasome inhibitor, carfilzomib, with a different mechanism of action was approved for pre-treated multiple myeloma patients. Developing second generation proteasomal inhibitors with improved properties or for different indications is the focus of many pharmaceutical companies (Petroski, 2008), as well as investigating of other clinical targets of the UPS. Although the proteasomal inhibitors are the only approved drugs acting on the UPS, a number of other molecules, affecting various classes of enzymes, including the E1 s and E3s, are currently under development and in anti-cancer clinical trials (Mattern et al., 2012). The rationale is to specifically target enzymatic activities or interactions responsible for pathological outcome to reduce side effects. Since blocking the proteasome is efficient in therapies, inhibiting the E1 enzyme activity could also be a viable therapeutic target (Xu et al., 2013; Yang et al., 2007). The strategies for inhibition of E1 activity involve interfering with ubiquitin-like protein adenylation, for example by blocking access of ATP, hindering E1 interaction with E2 or deterring the ubiquitin-like protein transfer to the E2. ATP-binding sites have been successfully targeted in the past in other types of enzymes, while inhibition of NEDD8 transfer was achieved with a small molecule inhibitor MLN4924, which inhibits NAE activity by producing a covalent adduct with NEDD8 after NAE-NEDD8 thioester formation (Brownell et al., 2010; Soucy et al., 2009). MLN4924 is currently in clinical trials for a variety of cancers. Targeting NEDD8 E1 instead of a ubiquitin E1 seems advantageous, since theoretically only one class of E3 ligases would be affected - the cullin-RING ligases, which are activated upon neddylation. However, this assumption will have to be verified, as neddylation is also involved in a large spectrum of biological functions and positive regulation of critical proteins, such as the tumour suppressor p53 (Tanaka et al., 2013; Xirodimas, 2008). Several other experimental E1 inhibitors exist, such as PYR-41 , which irreversibly binds to the active site of the ubiquitin E1 (Yang et al., 2007).

More selective inhibition could be achieved by blocking the activity of enzymes upstream in the ubiquitination cascade, such as the E2s and E3 ligases, and the deubiquitinating enzymes. The E2 conjugating enzymes appeared to be difficult targets because of the globular structure of the catalytic domain and the absence of a pronounced catalytic pocket (Nalepa et al., 2006). However, with the discovery of CC0651 , the first in class allosteric inhibitor of Cdc34 (also called Ube2R1 , an E2 involved in regulation of tumour suppressor p27 with a cognate SCF E3 ligase (Ceccarelli et al., 2011)), alternative ways of targeting this class of enzymes were proven viable (Edelmann et al., 2011). Currently, several molecules have been reported as E2 inhibitors, including Leucettamol A (Tsukamoto et al., 2006), Manadosterol's A and B (Ushiyama et al., 2012), NSC697923 (Pulvino al., 2012), Vitexin (Helms et al., 2012), CC0651 (Ceccarelli et al. 201 1) spectomycin B (Hirohama et al., 2013) and triazine analogues (Sanders et al., 2013)

Besides allosteric mechanisms, protein-protein interactions could be targeted between E2 and E3, and/or the substrate, providing highly selective inhibition of a particular ubiquitination reaction. Indeed, a number of molecules disrupting the E3-substrate interactions have been reported. The Mdm2-p53 interaction was one of the first targets, yielding several interesting molecules, with nutlin-3 and JNJ-26854165 now in clinical trials for cancer (Yuan et al., 201 1). Moreover, seven inhibitors of lAPs (Inhibitor of Apoptosis Proteins), another class of E3s involved in controlling tumour progression, are also in clinical trials (Mattern et al., 2012). The IAP inhibitors are mimetics of Smac, a natural IAP binder, which induces IAP autoubiquitination and degradation, triggering apoptosis. In addition to molecules

investigated in clinical trials, a large number and variety of E3 activity inhibitors are under experimental development and used as research tools for studying the biological functions of the E3s, especially the SCF complexes and Mdm2. Yet another interesting approach has been proposed for targeting specific substrates for ubiquitination and destruction, using artificial ubiquitin ligases. So far, Protacs (protein-targeting chimeric molecules) have been used to artificially recruit ubiquitin ligases to a few chosen protein targets (Sakamoto et al., 2001 , 2003).

Furthermore, deubiquitinating enzymes or DUBs are also considered as interesting targets in the UPS due to their implication in disease states (Nicholson et al., 2007), resulting from their role in modulating protein fate by removing conjugated ubiquitin from modified substrates. Upon DUB inhibition, oncogenic substrates may be degraded faster, with simultaneous upregulation of proapoptotic proteins, which has been shown to be the case for WP1 130 (Kapuria et al., 2010), and HBX41 , 108 (Colland et al., 2009), which blocked activity of several DUBs. Analogously, inhibition of a proteasome-associated DUB called Usp14 effectively increased proteasomal degradation of protein aggregates found in neurological disorders (Lee et al., 2010). Other specific inhibitors of selected DUBs have also been reported (Reverdy et al., 2012).

Research focusing on ubiquitin binding domains suggested interesting alternatives to regulation of the UPS, besides targeting the enzymatic reactions. Ubistatins were the first identified chemical compounds to bind to the K48 ubiquitin chains, thus preventing the recognition of modified substrates by the proteasome and their subsequent degradation (Verma et al., 2004). On the other hand, a recently published work described a method for targeting the ubiquitin-enzyme interface, by stabilisation of weak ubiquitin interactions with specific proteins using ubiquitin variants (Ernst et al., 2013). The highly conserved ubiquitin structure allows low affinity interactions with a high number of ubiquitin-interacting proteins, which contributes to the dynamic regulation within the UPS. Upon mutating determined residues on the ubiquitin surface, interactions with specific proteins can be enhanced, resulting in inhibition or activation of selected reactions. The UPS has a huge clinical potential, due to the number of potential target proteins and reactions, identified as druggable. The need for discovery of new chemical modulators to dissect molecular mechanisms of diseases, with the ultimate objective of developing new therapies, motivates development of novel methods for further UPS exploration and identification of inhibitors of specific reactions.

High-throughput investigation of the ubiquitination mechanisms

Despite efforts directed at understanding ubiquitination, only a limited number of

mechanisms have been elucidated for particular sets of enzymes and their cognate substrates. Questions remain regarding the E2-E3 cooperation for ubiquitination of a specific target protein, structural mechanisms governing the execution of ubiquitin conjugation and the type and function of resulting modifications.

One of the challenges is to identify the physiological E2-E3 pairs for a specific substrate. Indeed, the transient nature of these complexes due to the low affinity of interactions causes problems in the use of standard techniques (Wenzel et al., 201 1 b). Overexpression or depletion of chosen enzymes and monitoring of substrate degradation have been successful in some cases, however such approaches are limited by compensation mechanisms within the UPS and requires prior knowledge of the substrate. Probably the most fruitful high- throughput studies have been the yeast two-hybrid (Y2H) screens (Fields and Song, 1989), where chosen E2s and RING E3 interactions were tested in a genetic system (Markson et al., 2009; van Wijk et al., 2009). However, the Y2H approach is not free of limitations, including potential absence of additional cell- or tissue-specific factors or testing E3 interactions with free E2s and not with functionally relevant E2s charged with ubiquitin.

With a diversity of opportunities available, novel methods for high-throughput exploration of the UPS network are necessary. At the same time, demand has increased for small molecules targeting specific interactions, which would be used as new probes of biological and disease mechanisms and as a tool for controlling cellular activities. Importantly, taking into account the complexity and extent of the UPS, a systematic discovery of chemical modulators and elucidation of mechanisms of action on a large scale seems to be a suitable approach, which would be followed by focusing on a particular target or mechanism.

Existing high-throughput assays for the activity of the ubiquitinating enzymes are mostly based on ubiquitination assays, where the formation of an E1- or E2-ubiquitin thioester is assessed or the ubiquitin conjugation to a particular substrate is measured. The use of a labelled ubiquitin allows a direct detection, if the label is a fluorophore, or indirect detection, with ubiquitin binding domains or antibodies against ubiquitin. Detection appears to be an important issue, as traditional western blotting methods are far from high-throughput, and protein microarrays or microplates coated with target substrates are costly and involve relatively complicated procedures.

Therefore, an objective of the present invention was to develop an effective, time- and cost- efficient, high-throughput assay, suitable for screening for modulators of the UPS activities in a systems approach.

Materials and Methods

Recombinant proteins and reagents

Proteins and reagents were purchased as indicated in the Table 1.

Table 1 : Recombinant proteins and reagents used in the UPS-CONA experiments presented in the present application.

Constructs were obtained as indicated, and expressed and purified by J. Koszela in the Auer lab.

For recombinant proteins purified, chemically competent E. coli BL21 (DE3) (BioLabs) were transformed with the pE28a-LIC plasmid containing the 6xHis-tagged cDNAs under an IPTG-inducible T7 promoter. Colonies were selected for kanamycin resistance and grown in 5 mL of Terrific Broth supplemented with 50 mg/L kanamycin overnight at 37 °C. The following day, 1 L of Terrific Broth with appropriate antibiotic was inoculated with the overnight culture at 37 °C for 4 h until the Οϋβοο reached 0.8. Protein expression was induced with 1 mM IPTG for 3 h at 37 °C and cell pellets were collected and kept at -80 °C. For cell lysis, pellets were thawed and incubated on ice for 30 minutes in 30 mL of lysis buffer (50 mM sodium phosphate buffer pH 8, 300 mM NaCI, 10% glycerol, 10 mM imidazole, 10 mM β-mercaptoethanol, protease inhibitor cocktail (Roche) with 30 mg lysosyme (Sigma)) and sonicated 5x for 1 minute at 50% maximum power (Sonic VibraCell). The lysate was cleared by spinning for 30 minutes at 4 °C at 17000 rpm. For protein purification, the cleared lysate was incubated with 2-4 mL of 50% slurry Ni²⁺ NTA beads (Invitrogen) for 2 h at 4 °C, poured over a Biorad column and the beads with bound 6xHis tag-proteins were washed twice with wash buffer (lysis buffer with 20 mM imidazole). Finally, the 6xHis-tagged proteins were eluted in 8 fractions of 1 mL of elution buffer (lysis buffer with 250 mM imidazole). The fractions with the highest protein concentration were pooled together, dialysed overnight in 20 mM Hepes pH 7.9, 100 mM NaCI, 1 mM DTT and stored at -80 °C. Protein purity at each purification step was analysed by SDS-PAGE and visualised using Coomassie staining and anti-6xHis tag western blotting. The final sample was checked for purity on HPLC using RP-C4 column and the protein concentration was measured using absorbance at 280 nm. The yield was approximately 10 mg per litre of culture. Bead Preparation

For most experiments, nickel nitrilotriacetic acid (Ni²⁺NTA) agarose micro-beads (Life Technologies) were used. 4 of agarose beads (50% slurry) were used per well of a 384- well plate. Before use, beads were filtered with 100 μηι filters (cell strainers, BD Biosciences cat. n° 08-771-19) to obtain beads of homogenous size using Ni²⁺NTA binding buffer (0.3 M NaCI, 10 mM imidazole, pH 8) and briefly spun down at < 2500 rpm to remove excess of liquid.

Protein attachment to beads

Beads were incubated with a saturating quantity of 6xHis-tagged enzyme or protein substrate of interest in binding buffer for 2 hours at RT. Generally, 10 to 20-fold excess of protein was used, for example for one 384-well plate well, 100 pmol (1 of 100 μΜ solution) of active protein was incubated with 4 μΙ_ of 50% slurry Ni²⁺NTA agarose beads, with an estimated total 6.67 pmol loading capacity, in 500 μΙ_ of binding buffer. After incubation, beads were extensively washed with binding buffer. On-bead ubiquitination reaction

Beads were placed in 10 μΙ_ volumes into wells of black, flat bottom 384-well plate (Greiner) using enlarged (cut) pipette tip or large orifice tips (Rainin). Ubiquitination reactions were prepared in 10 μΙ_ of energy regeneration buffer containing protein components to obtain final concentrations in 20 μΙ_ volumes as indicated in Table 2 and added onto beads. The plate was imaged immediately for time-resolved experiments or after 1-6 hours of incubation, depending on the reaction.

Table 2: Final typical concentrations of the components used in the on-bead ubiquitination reaction. Depending on the experiment, E1 only, E1 and E2 or all three enzymes (E1 , E2, E3) were added to the reaction.

Imaging

Plates were scanned using the Perkin Elmer's Opera® High Content Screening System (further referred to as the Opera®) at 20x magnification (air lenses) at a confocal plane of 30μηι height (corresponding to the estimated agarose bead equatorial plane), binning 2, using the detection settings as indicated in Table 3. 10 to 63 (whole well) image views were taken per well, depending on the experiment. For experiments requiring statistical analysis of ring fluorescence, 35 (7x5) fields in the centre of the well were chosen for visualising around 100 beads, and a well sublayout with 20 % image field overlap was applied to allow image stitching. For time-resolved experiments, images were acquired every 20 to 60 minutes for one to 6 hours, depending on the experiment. Table 3: Excitation and detection parameters used for the on-bead ubiquitination

experiments on the Opera® for different fluorophores.

Data analysis

Images acquired with the PerkinElmer's Opera® High Content Screening System as .flex files were visualised using the PlateMontage script from the Acapella software (Perkin- Elmer) (binning 2) and further processed with ImageJ to generate bead profiles. For statistical analysis, 20% overlapped image fields were "stitched" together using the Acapella software and saved as .tiff files. The BeadEval software was used on the PS04 confocal bead scanner instrument computer for quantitative analysis providing the bead number, position, ring intensity and central area intensity, which were then compared and visualised using Excel.

Results

UPS-CONA is a novel method for monitoring ubiquitination in vitro

The term UPS-CONA, as used in the present application, refers to a Confocal Nanoscanning (CONA) technology as applied to the activities of various enzymes from the ubiquitin system. CONA was, originally established to measure molecular interactions of combinatorial chemical libraries with fluorescent targets on-bead (Hintersteiner et al., 2009, 2010, 2012; Meisner et al., 2009). The binding and subsequent enzymatic conjugation of fluorescently labelled ubiquitin or ubiquitin-like proteins to a substrate of interest immobilised on bead can be detected with confocal imaging (Figure 3). The appearance of a fluorescent "ring" is measured over time and quantified to assess specificity and activity of the enzymes.

Assessing activity of various classes of ubiquitinating enzymes with UPS-CONA

UPS-CONA allows dissection and monitoring of each enzymatic step in the ubiquitination cascade. In UPS-CONA, the enzyme or protein of interest from the activities of various enzymes from the ubiquitin system is attached to the bead and incubated with other ubiquitination reaction components in solution, including fluorescently labelled ubiquitin. The enzyme activity and reaction progress can be detected under a confocal microscope as formation of fluorescent rings.

Ubiquitin activation with E1

Ubiquitin activation with an E1 enzyme is the first step in the ubiquitination cascade. To visualise the ability of an E1 enzyme to form a thioester conjugate with ubiquitin, we attached a 6xHis-tagged human E1 enzyme (Ube1) to the nickel-NTA agarose beads and incubated with FITC-labelled ubiquitin in a buffered solution, in the absence or presence of ATP. As expected, in the absence of ATP, only background fluorescence was observed, while in the presence of ATP, intensively fluorescent rings formed and the relative intensity difference could be measured (Figure 4). E2 charging with ubiquitin

In the next step of the ubiquitination reaction, the E1 enzyme interacts with an E2 and transfers ubiquitin to the active site cysteine of the E2 in a trans-thioesterification reaction. To assess this reaction step, we attached a human E2 enzyme to the agarose beads via HIS-tag Ni²⁺-NTA and incubated with fluorescently labelled ubiquitin and E1 in solution with or without ATP. Fluorescent rings were detected in the presence of ATP, which confirmed that the fluorescent ubiquitin was charged onto the immobilised E2. We tested several human E2s for their ability to be charged with ubiquitin while on bead (Figure 5). Those included Ube2C (also termed UbcHIO), involved in cell cycle regulation; Ube2D2 (also termed UbcH5b), involved in regulation of p53, a tumour suppressor; Ube2L3 (also termed UbcH7), implicated in cell cycle regulation; Ube2M (also termed Ubc12), which is an E2 for Nedd8, a ubiquitin-like protein; Ube2R1 (also termed Cdc34), a cell cycle regulator; and Ube2U, a novel E2 with unknown function. Additional negative controls included beads with no protein attached and beads with a catalytically inactive E2 mutant (Ube2U C89A), which did not produce rings.

Ubiquitin transfer to a HECT E3 ligase

The transfer from an E2 enzyme to the substrate is mediated by an E3 ubiquitin ligase. Two major families of E3 ligases exist: the RING finger ligases, which provide a structural support for the E2-substrate interaction and the HECT ligases, which accept ubiquitin on their active site cysteine before transferring it to the substrate (Metzger et al., 2012). Here, we tested ubiquitin transfer to a HECT ligase, E6AP. E6AP was attached to the beads and incubated with ubiquitination reaction containing fluorescent ubiquitin, E1 (Ube1), E2 (Ube2L3), in the absence or presence of ATP (Figure 6). Fluorescent rings formed only in the presence of ATP, indicating that ubiquitin was conjugated onto the E3. Moreover, we confirmed that the ubiquitin-E3 thioester is sensitive to reducing conditions by adding DTT, which within minutes caused a decrease in ring intensity.

Ubiquitin conjugation to a substrate protein

We evaluated the assay suitability for monitoring more complex reactions. We tested p53, a tumour suppressor involved in DNA repair and apoptosis, as substrate for ubiquitination. The on-bead substrates were incubated with fluorescent ubiquitin, appropriate E1 , E2 and E3 enzymes, in the absence or presence of ATP. The fluorescent rings were detected in the presence of ATP, which indicates that all the enzymatic steps of the reactions occurred as expected and resulted in ubiquitin attachment onto p53 (Figure 7). The intensity of the rings formed on the on-bead p53 were visibly thinner and less intense as compared to the rings formed on an on-bead E1 or E2 in previously described experiments. A decreased intensity could be explained by the complexity of the reaction taking place and the expected autoubiquitination of Mdm2 (E3) in solution. The fluorescent ubiquitin present in the sample would in part be conjugated onto the in-solution Mdm2 and therefore not contribute to the p53 ubiquitin conjugation-dependent ring intensity. Monitoring the ubiquitin activation with E1 and transfer to an E2 enzyme

UPS-CONA allows detection of more than one reaction steps in a single well of a microplate. As a preliminary example, we chose to test ubiquitin transfer from Ube1 , a human E1 for ubiquitin, to the human E2 enzyme Cdc34 (Ube2R1). Ube1 was attached to the beads and incubated in the presence or absence of Cdc34 in solution. Reaction without ATP was used as additional control. With no Cdc34, we observed formation of fluorescent rings,

representing fluorescent ubiquitin charging onto the on-bead E1. When Cdc34 was present in the solution, fluorescent ubiquitin was homogenously distributed in the well, supposedly in conjugates with Cdc34 (Figure 8). UPS-CONA is sensitive to inhibitors of ubiquitination

Since the novel on-bead ubiquitination assay is suitable for monitoring ubiquitination and ubiquitination-like reactions step-by-step, we anticipated that the assay could be used for screening modulators of these reactions. Indeed, high-throughput screens would be possible due to the miniaturisation and multiplexing of the assay. As a proof of concept, we tested three known inhibitors of ubiquitination reactions: PYR-41 , an inhibitor of Ube1 (E1 for ubiquitin) (Figure 9); MLN4924, a NAE inhibitor (E1 for Nedd8) (Figure 10) and nutlin-3, which inhibits p53-Mdm2 interaction and subsequent p53 ubiquitination (Figure 11). PYR-41 inhibitory activity on Ube1 was evaluated based on the efficiency of Ube1 to transfer ubiquitin onto on-bead Cdc34. Decrease in ring intensity fluorescence corresponded to decrease in Ube1 activity in a concentration-dependent manner. Similarly, MLN4924 compound inhibited NAE ability to transfer Nedd8 to Ube2M. Finally, p53 ubiquitination levels were decreased as expected with increasing concentrations of nutlin-3. These results confirmed that UPS-CONA is sensitive to ubiquitination inhibitors, and can be adapted to high throughput screens for such inhibitors.

Time-resolved monitoring of ubiquitination

Ubiquitination is a dynamic process and different ubiquitinating enzymes, even from the same class, function with variable specificity and efficiency. UPS-CONA, due to its miniaturised setup, allows progressive monitoring of ubiquitination on a bead substrate, which is read as ring intensity variation over time. Using a high-content confocal microscope such as the Opera®, multiple time points can be measured, limited only by scanning speed, which depends on the number and length of exposures, number of wells and fluorophore stability. To illustrate a possible outcome of a time-course experiment, we observed ubiquitin charging onto Cdc34 (Ube2R1) and measured the maximal ring intensity every hour. The fluorescence intensity of the rings, corresponding to the number of fluorescent ubiquitin molecules charged onto Cdc34, quickly increased in a linear manner during the first hour, then increased at a lower rate to peak at 4 hours of incubation and finally decreased slightly as a possible result of re-equilibration or fluorophore bleaching (Figure 12). Quantification and statistical analysis of several beads per well in this format allows robust conclusions to be drawn. Multi-colour ubiquitination assay

The multi-colour setup of UPS-CONA is a basis for quantification of the ubiquitin-to-substrate ratio and for simultaneous monitoring of ubiquitination rates of various enzymes. Indeed, the use of 1 : 1 -labelled proteins would allow the assessment of ubiquitination dynamics over time and to determine the type of ubiquitination (mono- versus poly-ubiqutination, and/or linkage specificities with differentially labelled mutant forms of ubiquitin). We confirmed that fluorescent ubiquitin can be charged onto a chemically labelled Ube1 (AlexaFluor-633) and onto a GFP fusion of Cdc34 could be detected using the Opera® (Figure 13 and Figure 14).

Detection of different bead populations in one well

Due to the miniaturisation to a single micro-bead level combined with multi-colour detection, UPS-CONA can be used for multiplexed high-throughput screens. Existing high-throughput technologies use microplate to limit the volume and amount of components required for each reaction. In UPS-CONA, however, around 200 micro-beads can be screened in a single well of a 384-well plate, which provides a large number of technical replicates. Furthermore, fluorescent labelling of several proteins of interest with various fluorophores allows simultaneous monitoring of a set of enzymes or of multiple steps of a reaction in one well. It is currently possible to detect three different colours using the Opera® system, as described in the Materials and Methods. Figure 15 shows detection of two different bead populations, prepared separately and then mixed together in one well: beads with attached Ube1 labelled with AlexaFluor-633 and beads with AlexaFluor 488-labelled Cdc34. With a multi-colour protein labelling approach and the actual fluorescence detection possibilities on the Opera®, it is possible to monitor or screen for inhibitors of up to three independent reactions per well with replicates, as illustrated in the Figure 15. Considering the use of 384- or possibly even 1536-well plates, multi-colour detection and single-bead miniaturisation, UPS-CONA is thus demonstrated as an ultra-high-throughput method. Quantification

The UPS-CONA multi-colour fluorescence setup combined with high-resolution detection methods allows a precise, quantitative analysis of the results. The results presented so far are of a qualitative nature. We were able to quantitatively analyse an exemplary experiment of a fluorescent (TMR fluorophore, 555 nm) 6xHis peptide loading onto TentaGel beads adapted for his-tagged protein binding via Ni²⁺NTA. Beads were screened on the Opera® system and the images were analysed using the BeadEval script. For each well, the software generated a well picture with contours of beads identified (Figure 16) and a list of parameters (bead number, radius, ring and central area intensity), which then were represented in charts. Ring versus centre intensity plot allows visualisation of the correlation between these two parameters and identification of "outliers" - beads with internal fluorescence, which would be ignored in further analysis as experimental artefact. The ring intensity of beads within a well follows approximately a normal distribution, as represented in the second chart (Figure 16). Discussion

UPS-CONA is suitable for detection of ubiquitin charging onto various enzymes from the ubiquitination pathway (E1 activation, E2 charging, E3 transfer) and ubiquitin conjugation onto a protein substrate, as we showed with exemplary experiments summarised in the Table 4. Table 4

The assay is applicable for reactions with other ULPs, including neddylation, SUMOylation, ISGylation and others. UPS-CONA is also inherently suitable for investigation of the activity of the deubiquitinating enzymes (DUBs), which can be provided in soluble form to act on a pre-loaded ubiquitin-substrate conjugate. Protein-protein and protein-compound interactions of fluorescently labelled molecules can also be detected with UPS-CONA, for example by Forster Resonance Energy Transfer (FRET) between fluors of appropriate emission and excitation wavelengths.

Automation for high-throughput screening

The inventors have shown that UPS-CONA is sensitive to known ubiquitination inhibitors. An automated setup would be optimised for high-throughput applications. A liquid-handling robot (Biomek 2000), has been successfully tested for distribution and mixing together 1) 10 μΙ_ of beads with attached protein, 2) 2 μΙ_ of control compounds in 50% DMSO, for a final DMSO concentration of 2.5%, 3) the ubiquitination reaction mix, from 96-well plates onto a 384-well plate in only 10 minutes. Figure 17 shows a representation of a general workflow for an automated high-throughput screening. Quantitation

For quantification of the results observed on the Opera®, the following optimisations can be applied:

- Use of 1 : 1 fluorescently labelled enzymes and substrates

- Homogenous size and shape of micro-beads (best for bead recognition and parameters standardisation)

- Establishment of the relationship between detected fluorescence intensity and the

number of molecules - Script adaptation for high-throughput analysis on the Opera®.

Optimisation of quantitative aspects allows for production of data on ubiquitination reactions kinetics over time. Screening options

Two major applications of the UPS-CONA system include addressing biochemical questions regarding the ubiquitin system and screening for modulators of ubiquitination reactions. Due to the modularity, flexibility, miniaturisation and other features of the assay, various screening formats are available, as well as a large choice of library formats suitable for screening. Moreover, multiple types of biological questions can be asked, reflecting the target and disease focus of a research group or company, which can be also extended to investigations of the UPS in other species.

1 ) Screening formats

Various screening formats are available via UPS-CONA as described below. The flexibility of the setup is ensured by the possibility of multiplexing due to the single micro-bead, multicolour detection method. Examples of screening formats available in UPS-CONA.:

1. Description: Screening for modulators of one type of reaction per plate well using

chemical libraries. Example: Screening for p53 ubiquitination inhibitors: p53 is linked to beads, suitable ubiquitination components are present in the medium, and different compound sub-library is provided to each well. This allows for screening for library members which affect ubiquitination of p53 at various possible steps.

2. Description: One type of reaction per well (or set of wells), and several different

reactions representing stages of a ubiquitination pathway of interest. This can be used, for example, for dissecting the mechanism of inhibition of ubiquitination of a protein by a test agent. Example: In each well column of a plate a different step of the p53 ubiquitination pathway is set up, and assessed in the presence of inhibitors of the complete ubiquitination reaction. The specific inhibitory activity of the inhibitor (e.g. its target and the part of the pathway which it effects) can thus be accurately determined.

3. Description: Multiple reactions in a single well. Example: Screening for inhibitors of ubiquitination of three differentially labelled different target proteins in a single well allows to screen for inhibitors of ubiquitination of various targets simultaneously to identify ubiquitin preference of various E2 enzymes using different coloured labels for each type of ubiquitin moiety.

4. Description: UPS-CONA, followed by bead-picking and mass spectrometry. Example:

Screening for inhibitors of ubiquitination of different target proteins. One target protein can be attached on suitable beads; beads can be mixed to add ~ 400 beads per well of a 384 well plate. In this embodiment of the invention, each target contains the same dye or fluorescent protein. E.g. if all 38 E2 conjugating enzymes of the human genome are linked to beads in a blue fluorescent protein conjugated set-up, each targets is represented ~ 10 times in a 384 well plate format. A specific E1 , ATP and ubiquitin labelled with a red dye are added including one or a mix of small molecule or

peptidomimetics library compounds. If one compound or a mix of compounds inhibits the ubiquitination of a particular E2 or several E2s, these beads will not show fluorescence ring formation after a certain incubation time. These beads are picked by a bead picker, and subjected to proteomics analysis to identify the E2 which was, against control, not ubiquinylated.

2) Biological and UPS mechanistic questions

UPS-CONA can be used for the following, exemplary but not exhaustive, list of UPS-related investigations:

• Monitoring each step of the ubiquitination/ubiquitination-like reaction:

On-bead: E1 , E2, E3, auxiliary proteins or substrate

o Other ubiquitination components in solution

• Deubiquitinating activity of DUBs and their specificity

o Different ubiquitin chain types on-bead, labelled differentially, and monitoring the deubiquitinating reaction kinetics

• Monitoring several reaction steps in one well:

o On-bead and in-solution enzymes labelled with different fluorophores

• Comparing the ubiquitin/ubiquitin-like protein conjugation rate of several enzymes from the same class

o For example, each well would contain beads with different E2s (one or up to

three different per well) and the ubiquitin-charging rate will be compared between the wells/beads

• Comparing the ubiquitin/ubiquitin-like protein conjugation rate of enzymes from different classes

o To investigate the reaction kinetics of each step of a complex reaction

• Investigation of E2 preferences for any E3 of interest

• Investigation of the preferred chain type generated on a substrate by various E2/E3s

• Screening for optimal enzyme sets for a substrate of interest

o On-bead substrate incubated with different enzyme sets (E2/E3 pairs) in solution; reaction kinetics will reveal the optimal set in vitro • Investigating the ubiquitination of a substrate of interest by proteins present in a cell lysate

o An on-bead substrate of interest (purified or from lysate) is incubated with lysates from cells treated under different conditions (for example, from different phase of the cell cycle, upon drug treatment, different cell lines etc.. In the presence of purified or expressed fluorescent ubiquitin, the kinetics of the substrate ubiquitination can be evaluated.

3) Disease and target focus

Since UPS perturbations are the cause of various diseases, most of the ubiquitinating and deubiquitinating enzymes, activity of which can be evaluated with the present assay, have clinical impact.

4) Expansion to other species

The UPS is conserved amongst all eukaryotes, from single cell organisms to humans. The importance of ubiquitination in regulating protein degradation and many other cellular processes makes the UPS a sensitive and large target, which can be attacked at various points. Therefore, a targeted deregulation of the UPS in human and livestock pathogens is a potentially valid therapeutic approach against infectious diseases. Additionally, the interest has been rising regarding the molecular interactions between the host and microbial/parasite proteins. In particular, a growing literature elaborates on the components of the parasite UPS (Mizushima et al., 2008, Hashimoto et al., 2010) or viral proteins (Engel 2013;

Viswanathan 2010; Marvin and Wiethoff, 2012; Fuchs 2012) interfering with the host immune system. The present invention can be used to investigate the host-pathogen protein interactions and enzymatic activities and to screen for molecular agents, which would abolish pathological reactions. If such agents present drug-like structures, they could be considered as potential leads for novel treatments against infectious diseases, such as Shigella, HIV, influenza, malaria or Chagas disease. Further Exemplifications of Embodiments of the Invention

1 UPS-CONA as a method for investigation of compound specificity within the ubiquitination system and for elimination of false positives Summary

From a UPS-CONA screen for inhibitors of ubiquitination of Ube2C, which is an

acknowledged cancer target, two potential hits were identified and confirmed using alternative methods. UPS-CONA was used to assay the specificity of the hits and to perform a structure-activity relationship analysis with a set of analogues. Additionally, the two-colour ratio method allowed us to eliminate false positives. Materials and Methods

Proteins: 10xHis-eGFP-Ube2C and 10xHis-eGFP in pRSET vector were expressed in BL21 (D3) E. coli and purified using the AKTA purification system on a HiTrap cobalt column. 6xHis-Cdc34, 6xHisUbe2L3, 6xHis-Ube1 in pET28a-LIC vectors were expressed in

BL21 (D3) E.coli and purified using standard NiNTA agarose protocol. Protein purity was verified by HPLC and SDS-PAGE. Enzymatic activities were confirmed by UPS-CONA and in standard gel-based ubiquitination assays. Flag-Ube1 and WT ubiquitin were purchased from Sigma and BostonBiochem, respectively. CysO-ubiquitin was acquired from the Sicheri lab and labelled with Cy5-maleimide, producing Cy5-ubiquitin.

UPS-CONA:

NiNTA agarose beads were sieved through 100 μηι and 120 μηι filters for improved size homogeneity and 1.5 μΙ_ of beads (40% slurry) were incubated with 10 pmoles of the required His-tagged protein per well of a 384-well plate for 30 minutes at 4°C. 10 μΙ_ of bead suspension per well was distributed in the wells and the compounds (20 μΜ) were added prior to adding a ubiquitination reaction mix (0.3 μΜ Cy5-ubiquitin, 0.1 μΜ E1 , 5 mM ATP in energy regeneration buffer). The plate was incubated for 2 h at room temperature and scanned on the Opera™ HCS instrument (PE) or on the PS04 confocal microspectroscopy systems. The data was analysed using Acapella scripts (PE), ImageJ and Excel.

Results

The UPS-CONA assay was optimised for screening for inhibitors of Ube2C activity (Figure 18). In brief, 10xHis-eGFP-Ube2C active fusion protein was immobilised on NiNTA agarose beads and incubated with fluorescently labelled Cy5-ubiquitin under conditions allowing the ubiquitination reaction to occur. Ubiquitin charging was detected on confocal microscope in the form of fluorescent rings in the Cy5 channel, corresponding to Cy5-ubiquitin conjugated to the on-bead Ube2C. Inhibition of the reaction was expected to be detected as a decrease in the ring intensity ratio between Cy5-ubiquitin and eGFP-Ube2C. A 2112-member allosteric discovery library from a strategic partner company was screened in batches of 11 compounds prior to hit deconvolution and identification of individual hits from active pools. Two compounds (Hit1 and Hit2) had a detectable effect on the ring intensities in the Cy5 channel. The two potential hits were tested for specificity towards Ube2C versus other E2s and an E1 enzyme. 108 analogues were received from our collaborator and assessed for activity in inhibiting Ube2C.

Confirmation of Hit1 and Hit2 inhibitory effects on Ube2C activity

Two primary hit compounds identified in the mixture screen as inhibitors of ubiquitin charging onto the on-bead conjugated Ube2C were confirmed individually using UPS-CONA. The reduction of the Cy5-ubiquitin signal in the rings formed around the on-bead conjugated 10xHis-eGFP-Ube2C in the presence of Hit1 and Hit2 was confirmed (Figure 19). Moreover, the inhibitory effects were confirmed in a UPS-CONA assay using bead immobilised non- fluorescent 6xHis-Ube2C (Figure 19) and in a standard gel-based ubiquitination assay.

Hit1 and Hit 2 are not specific to Ube2C

Hit1 and Hit2 were tested for ubiquitination activity of Ube1 (E1) and two different E2s:

Ube2L3 and Ube2R1 by UPS-CONA. A reduction in the Cy5-ubiquitin signal conjugated to the on-bead E1 or E2s was detected in all experiments (Figure 20). The observed inhibition of the E2 activities can be explained by inhibition of the E1 enzyme, which acts upstream in the ubiquitination reaction. To confirm the inhibitory effect of Hit1 and Hit2 on Ube1 , the E1- ubiquitin charging reaction was performed with a range of compound concentrations in a gel- based assay. Both Hit1 and Hit2 indeed inhibited the Ube1-ubiquitin thioester formation. Additionally, Hit1 and Hit2 seemed to affect physical properties of most of the tested proteins, including BSA, by causing a smearing effect on SDS-PAGE. It is therefore likely that the compounds are not specific inhibitors of Ube2C ubiquitination.

SAR with 108 analogues

In search of specific Ube2C inhibitors, 108 scaffold analogues of the initial hits were screened twice by UPS-CONA with on-bead eGFP-Ube2C and Cy5-ubiquitin in a

ubiquitination reaction as described in the methods section. We were looking for compounds with a minor intensity decrease of eGFP-Ube2C and a strong intensity decrease of Cy5 signal from conjugated ubiquitin.

However, the compounds which did not significantly affect the eGFP signal, did not affect the Cy5- ubiquitin signal either (Figure 21). Conclusion

The experimental data described above confirm that UPS-CONA can be successfully applied to screen for inhibitors of ubiquitination reactions, as inhibition of the reaction by initially identified hits was confirmed with SDS-PAGE as an alternative method which is not fluorescence dependent. UPS-CONA can be used to investigate compound specificity towards other ubiquitination reactions in a quick and reliable way. Finally, most of false positive hits which interfere with the fluorescent assay setup can be successfully identified using the two-colour ratio approach between the on-bead protein and the ubiquitin conjugate.

2 Assaying autoubiquitination of CHIP E3 ligase

Introduction

CHIP is a HECT-type E3 ubiquitin ligase, involved in regulation of activity of molecular chaperones such as Hsp70, Hsc70 and Hsp90. CHIP regulates inflammatory immune responses and therefore is a potentially relevant target in autoimmune disease, infection and cancer (Chen et al., 2013).

HECT E3 ligases possess an active site cysteine, to which ubiquitin is transferred from a cognate E2 enzyme in the ubiquitination cascade. Additionally, CHIP is able to

autoubiquitinate itself, by transferring the ubiquitin thioester from the active site to specific lysines in its sequence. Ubiquitination of CHIP alters its activity.

Previously, conditions for activity of other HECT E3 ligases have been successfully optimized in UPS- CONA. For CHIP, both ubiquitin charging on active site and

autoubiquitination were expected to be detected. In this report, the optimization process for detection of autoubiquitination of CHIP is reported.

Materials and Methods

Proteins: 6xHis-CHIP, untagged CHIP, 6xHis-UbcH5a and untagged UbcH5a were received from Kathryn Ball's lab (UoE). Fluorescently labelled Cy5-ubiquitin (Cy5-Ub) was prepared. Ube1 was purchased from Sigma-Aldrich. UPS-CONA: NiNTA agarose beads (Qiagen) were incubated with 6xHis-tagged substrate in amounts referred to in the experimental part, washed and placed in wells of a clear flat bottom 384-well plate (MMI). Ubiquitination reaction mix was added (energy regeneration buffer:50 mM Tris-HCI, pH 7.5, 5 mM MgCI2, 10 mM creatine phosphate, 3.5 U/mL creatine phosphokinase; 0-0.3 μΜ Cy5-Ub, 0-0.2 μΜ Ube1 , 0-0.25 μΜ UbcH5a, 5 mM ATP as indicated) and incubated for 1 h at room temperature. The beads were visualised on the Opera™ confocal high-content imaging system (Perkin-Elmer) set up for Cy5 detection. Images were analysed using the Acapella software (Perkin-Elmer) to generate

representative images of beads from each well, ImageJ to analyse bead intensity profiles and Excel.

Results

Test of CHIP activity by UPS-CON A

Prior to a larger scale optimisation, a quick activity test was performed to confirm the autoubiquitination activity of CHIP on bead. Controls included blank beads (no CHIP), beads with conjugated CHIP, and with 0.3 μΜ Cy5-Ub only, 0.3 μΜ Cy5-Ub and 0.15 μΜ Ube1 only, 0.3 μΜ Cy5- Ub, 0.15 μΜ Ube1 and 0.2 μΜ UbcH5a without ATP (Figure 22). All control experiments were negative, and the sample well containing on-bead CHIP, Cy5-Ub, Ube1 , UbcH5a showed detectable signal from ubiquitination of the on-bead conjugated CHIP within minutes after adding ATP. Addition of DTT, which removes the ubiquitin thioester formed on the active site cysteine, did not detectably affect the ubiquitination signal. This suggests that the signal came mostly from modification of CHIP by ubiquitin through its autoubiquitination and not from the formation of a thioester on the active site of CHIP.

Optimisation of amount of substrate needed on bead

For the development of a highly sensitive autoubiquitination assay the amount of CHIP conjugated to test beads in each well needed to be optimized. For detecting minimal CHIP autoubiquitination activity CHIP autoubiquitination and UbcH5a ubiquitin charging reactions with 0-20 pmoles of protein per well were analysed (Figure 23). The optimal amount of the substrate protein per well was determined to be 15 pmoles.

Optimisation of E1 concentration

To optimise the concentration of E1 (in solution), concentrations ranging from 0 to 0.25 μΜ of Ube1 were tested. The strongest signal was obtained with 0.15 μΜ of Ube1 (Figure 24).

Optimisation of E2 concentration

Similarly, to optimise the concentration of E2 (in solution), concentrations ranging from 0 to 0.2 μΜ of UbcH5a/E2D1 were tested. The strongest signal was obtained with 0.2 μΜ of UbcH5a (Figure 25). Further assay optimization might be achieved with higher concentrations of UbcH5a.

Optimisation of ubiquitin concentration

Finally, to optimise the concentration of ubiquitin (in solution), concentrations ranging from 0 to 0.3 μΜ of Cy5-Ub were tested. The ring intensity did not vary significantly between 0.1 and 0.3 μΜ, however the best signal to background ratio at 1 hour reaction was observed with 0.2 μΜ of Ub (Figure 26). Conclusion

CHIP, a clinically relevant E3 ligase was confirmed to work as target in the on-bead ubiquitination assay. The optimized conditions will be used for screening for modulators of the CHIP activity. 3 Parallel monitoring of ubiquitination-like reactions

Summary

Described is an example of one new feature of UPS-CONA, monitoring ubiquitination and neddylation in parallel in the same well. The method can be used to test specificity of reactions towards a substrate, specificity of enzymes, and chemical entities towards multiple ubiquitination-like reactions, such as ubiquitination, neddylation, sumoylation, ATGylation, FATIOylation etc.

Materials and Methods

6xHis-Cdc34 and 6xHis-Ubc12 were purified in the Auer lab on NiNTA agarose beads

(Qiagen) and quality was checked by SDS-PAGE and HPLC. Activity was confirmed in gel- based ubiquitination (Cdc34) and neddylation (Ubc12) assays, as well as on bead (UPS- CONA). Cy5-Ubiquitin was produced in the Auer lab. FITC-NEDD8 was purchased from BostonBiochem (cat. n. UL-830), Flag-Ube1 from Sigma Aldrich (cat. n. SRP0440), NAE (APPBP1/Uba3) from BostonBiochem (cat. n. E-313).

20 picomoles of 6xHis-Cdc34 or 6xHis-Ubc12 per well were incubated with 1.5 μΙ_ of 40% slurry sieved 100-120 μηι NiNTA agarose beads. After binding, the beads were extensively washed, placed onto 384-well plate (MMI) and incubated with ubiquitination/neddylation mix (300 nM Cy5-Ub, 300 nM FITC-Nedd8, 100 nM NAE, 100 nM Ube1 , 5 mM ATP) for 1 h at room temperature. The plate was imaged on the Opera™ HCS instrument (Perkin-Elmer) using 488 and 640 nm excitation (for FITC and Cy5, respectively). Results

The goal was to investigate whether the UPS-CON A setup presents an opportunity for monitoring in parallel more than one ubiquitination-like reaction in a single well of a test plate. Cdc34, which is exclusively a ubiquitinating E2, and Ubc12, which is exclusively a neddylating E2, were immobilized on beads and placed in separate wells. The ubiquitination- neddylation mix was then added to the beads, containing fluorescently labelled Cy5- Ubiquitin, FITC-Nedd8 and non-labelled E1 enzymes for ubiquitin and Nedd8 (Ube1 and NAE, respectively). Upon addition of the reaction buffer containing ATP, the reaction results were imaged on the Opera and represented in Figure 27 (left panel). The ubiquitination rings, detected through 640 nm excitation of Cy5-ubiquitin, appeared only in the well with on- bead Cdc34, while the neddylation rings, detected through 488 nm excitation of FITC- Nedd8, were formed only in the well with on-bead Ubc12, as expected. Conclusion

The experiment described above showed that UPS-CONA is suitable for monitoring more than one possible ubiquitination-like reaction in a well. No cross- reactivity towards non- cognate E2 enzymes was observed. The system can be extended to having multiple target proteins on bead in a single well, by adding a distinguishable fluorescent label as illustrated by green and red on the right side of Figure 27, or using a post-reaction identification of on- bead substrate (mass spectrometry, FACS). Ubiquitination- like reactions may include neddylation, sumoylation ATGylation, FATI Oylation etc., which makes this approach interesting for investigation of cross-reactivity between various cascades and for testing modulators of such reactions and their specificity (including inhibitors, enhancers, specificity modifiers etc.).

4 Three colour E2 assay system for UPS-CONA

Summary

In preparation for a multiplexed screening layout, three different E2 ubiquitin conjugating enzymes with differentiable fluorophores were imaged by confocal scanning and identified in a single well.

Materials and Methods

10xHis-mTurq2-Ube2K, 10xHis-eGFP-Ube2R1 (also called Cdc34) and 10xHis-E2-Crimson- Ube2L3 were cloned, expressed and purified in the Auer lab. Purity was assessed by SDS- PAGE and HPLC, and described in a separate report. The following excitation/emission settings were used to visualize fluorescent protein conjugate binding events via fluorescence emission halos on the Opera™ HCS instrument (Perkin-Elmer): mTurq2: 445 nm laser, 475/40 nm detection filter; eGFP: 488 nm laser, 525/30 nm detection filter; E2-Crimson: 561 nm laser, 660/150 nm detection filter. The settings are listed in Table 5 below.

Table 5: Excitation and emission settings used on the Opera HCS (Perkin-Elmer) to detect four fluorophores.

*Cy5-Ubiquitin was not tested in this experiment, the settings are listed for reference.

Results

For uHTS applications, more than one on-bead substrate protein needs to be placed in a single well of a screening plate. The different substrates need to be optically resolved without significant interference. As an example for a triple-target screening design using the UPS-CONA technology, we tested three different E2 ubiquitin conjugating enzymes: Ube2K, Ube2R1 (Cdc34) and Ube2L3. Each of these E2s is fused to a different fluorescent protein: mTurquoise 2 (mTurq2), eGFP and E2-Crimson, respectively, of which detection specifics are indicated in Table 5. After separate incubations with NiNTA agarose beads, to which the proteins are bound via a target conjugated His-tag, the single E2 protein conjugated beads were mixed and placed in one single well. The Opera™ HCS (Perkin-Elmer) settings listed in the table above allowed to distinguish the three batches of beads and to decode the bound E2 using the colour code: mTurq2 was detected using 445 nm excitation, eGFP - 488 nm, and E2-Crimson - 561 nm (Figure 28). A weak signal leakage was observed, as mTurq2 rings were visible upon 488nm excitation. Since the leaking signal is weaker than the eGFP signal and weaker than the mTurq2 signal in the 445 nm channel, this issue can be resolved by applying corrections in data analysis. Conclusion

UPS-CONA allows for simultaneous detection of at least three on-bead substrate proteins and conjugates, such as ubiquitin. The multi-colour setup increases the assay throughput and allows testing in parallel specificity of potential modulators of the monitored binding and/or enzymatic reaction (activators, inhibitors, specificity modulators).

Concluding remarks

In conclusion, the UPS-CONA assay presents significant advantages listed below over existing in vitro approaches for studying and screening for modulators of the ubiquitination reactions:

- Suitable for monitoring in vitro ubiquitination and ubiquitination-like reactions (e.g.

Nedd8, SUMO, etc.)

- Modular: allows to dissect each step of the reaction, suitable for various classes of

ubiquitinating enzymes and various types of reaction

- Sensitive to inhibitors: suitable for screening for inhibitors.

- Time-resolved: reaction dynamics over time.

- Miniaturised & high-throughput:

o Multiple technical replicates per well - due to miniaturisation to a single micro- bead level

o Simultaneous monitoring of various reactions per well - ensured by a multi-colour fluorescence setup

o High sensitivity detection

o Semi-automated and simple: no washing or additional secondary detection steps, only mix-and-read procedure.

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Claims

Claims

1. A method for investigating molecular interactions and enzyme activities related to the ubiquitin-proteasome system (UPS), the method comprising:

a) providing at least one surface with a target moiety bound to at least a portion thereof;

b) i) exposing said surface to a medium comprising a labelled ubiquitin moiety and a component or components of the ubiquitination system suitable to conjugate the ubiquitin moiety to a target moiety; and/or

ii) wherein said target moiety is conjugated to a labelled ubiquitin moiety exposing said at least one surface to a medium comprising a deubiquitinating enzyme (DUB) suitable to remove the ubiquitin moiety from a target moiety; c) providing conditions permissive for the conjugation/removal of the labelled ubiquitin moiety to/from the target moiety; and

d) detecting conjugation and/or removal of the labelled ubiquitin moiety by

detecting labelled ubiquitin moiety located at or near the surface.

2. The method according to claim 1 , wherein the surface is the surface of a substrate and the substrate comprises one or more particles and the surface is the outer surface of the particles.

3. The method according to claim 2, wherein the surface is the outer surface of one or more beads.

4. The method according to claim 3, wherein the one or more beads are substantially spherical.

5. The method according to any one preceding claim, wherein a plurality of surfaces are provided.

6. The method according to any one preceding claim, wherein the method is used for detecting conjugation of a ubiquitin moiety to a target moiety.

7. The method according to any one of claims 1 to 5, wherein the method is used for detecting the ability of a test agent to modulate conjugation of a ubiquitin moiety to the target moiety or removal of a ubiquitin moiety from the target moiety.

8. The method according to any one preceding claim, wherein the method comprises providing a test agent, and measuring the effects of the test agent on the rate or extent of ubiquitin moiety conjugation/removal.

9. The method according to claim 8, wherein the test agent is any suitable agent, including, chemical compositions, such as small molecules, peptides, proteins, and nucleic acids.

10. The method according to any one preceding claim, wherein the target moiety is any moiety which is susceptible to conjugation to a ubiquitin moiety, or which is to be tested for its ability to be conjugated to a ubiquitin moiety.

1 1. The method according to claim 10, wherein the target moiety is a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), a ubiquitin ligase (E3), or is a substrate or putative substrate of ubiquitination.

12. The method according to any one of claims 2 to 11 , wherein the particles have a major dimension of from 1 to 1000 μηι.

13. The method according to claim 12, wherein the particles have a major dimension of from 20 to 250 μι ι.

14. The method according to any one of claims 2 to 13, wherein the particles have a substantially uniform size distribution.

15. The method according to any one preceding claim, wherein the ubiquitin moiety is labelled with a fluorescent label.

16. The method according to any one preceding claim, wherein the target moiety is labelled.

17. The method according to any one preceding claim, wherein the method comprises monitoring multiple reactions in a single vessel.

18. The method according to any one preceding claim, wherein the method comprises using multiple fluorescent labels which emit light at different wavelengths, and are therefore distinguishable from each other.

19. The method according to any one preceding claim, wherein the method comprises measuring the progress of conjugation or removal of the labelled ubiquitin moiety over a period of time.

20. The method according to any one preceding claim, wherein the method is a method for screening for modulators of ubiquitination or deubiquitination of a substrate protein, the method comprising providing said substrate protein linked to a surface, and observing for effects of a test agent upon ubiquitination or deubiquitination.

21. The method according to any one of claims 1 to 19, wherein the method is a method for investigating details of the mechanism of ubiquitination or deubiquitination of a substrate protein.

22. The method according to any one of claims 1 to 19, wherein the method is a method for determining which steps of the ubiquitination or deubiquitination pathway a test agent modulates.

23. The method according to claim 22, wherein the method is a method for determining the specific target of the test agent.

24. The method according to any one preceding claim, wherein the kinetics of various enzymes of the ubiquitination or deubiquitination pathway are studied.

25. The method according to any one preceding claim, wherein a plurality of target

moieties are each labelled with a different and distinguishable fluorophore and a ubiquitin moiety is labelled with yet another different and distinguishable fluorophore.

26. The method according to any one preceding claim, wherein several different ubiquitin moieties are labelled with different and distinguishable fluorophores.

27. The method according to any one preceding claim, wherein the at least one surface comprises a plurality of different target moieties and the method further comprises the step of e) analysing the plurality of different target moieties on the at least one surface to determine the identity of the plurality of different target moieties.

28. The method according to claim 27, wherein the identity of the plurality of different target moieties is determined by analytical methods including, mass spectrometry, high performance liquid chromatography (HPLC), gas chromatography (GC), and immunoblotting.

29. The method according to any one preceding claim, wherein the method is a screen for identifying one or more compounds for treatment of a disease.

30. An assay for investigating molecular interactions and enzyme activities related to the ubiquitin-proteasome system (UPS), the assay comprising:

at least one surface having a target moiety bound to at least a portion thereof; and

at least one medium comprising a labelled ubiquitin moiety and a component or components of the ubiquitination reaction suitable to conjugate the labelled ubiquitin moiety to the target moiety; and/or

at least one medium comprising a deubiquitinating enzyme (DUB) suitable to remove a labelled ubiquitin moiety provided on the target moiety.

31. The assay according to claim 30, wherein the surface is a surface of a particle.

32. The assay according to either one of claim 30 or 31 , wherein the target moiety is a component of the UPS.

33. The assay according to either one of claim 30 or 31 , wherein the target moiety is a substrate (e.g. a protein) which is a known or putative target of ubiquitination in vivo.

34. An assay kit for investigating molecular interactions and enzyme activities related to the UPS, the assay kit comprising:

at least one surface having a target moiety bound to at least a portion thereof or adapted for convenient binding of a target moiety bound to at least a portion thereof; and

at least one medium comprising a labelled ubiquitin moiety; and

at least one medium comprising a component or components of the ubiquitination system suitable to conjugate the labelled ubiquitin moiety to the target moiety; and/or

at least one medium comprising a deubiquitinating enzyme (DUB) suitable to remove a labelled ubiquitin moiety provided on the target moiety.

35. The assay kit according to claim 34, wherein the surface is a surface of a particle.