Chemistry-Based Functional Proteomics: Mechanism-Based
Activity-Profiling Tools for Ubiquitin and Ubiquitin-like Specific
Proteases
Joris Hemelaar, Paul J. Galardy, Anna Borodovsky, Benedikt M. Kessler, Hidde L. Ploegh, and
Huib Ovaa*
Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115
Received November 21, 2003
Determining the biological function of newly discovered gene products requires the development of
novel functional approaches. To facilitate this task, recent developments in proteomics include small
molecular probes that target proteolytic enzyme families including serine, threonine, and cysteine
proteases. For the families of ubiquitin (Ub) and ubiquitin-like (UBL)-specific proteases, such tools were
lacking until recently. Here, we review the advances made in the development of protein-based active
site-directed probes that target proteases specific for ubiquitin and ubiquitin-like proteins. Such probes
were applied successfully to discover and characterize novel Ub/UBL-specific proteases. Ub/UBL
processing and deconjugation are performed by a diverse set of proteases belonging to several different
enzyme families, including members of the ovarian tumor domain (OTU) protease family. A further
definition of this family of enzymes will benefit from a directed chemical proteomics approach. Some
of the Ub/UBL-specific proteases react with multiple Ub/UBLs and members of the same protease family
can recognize multiple Ub/UBLs, underscoring the need for tools that appropriately address enzyme
specificity.
Keywords: ubiquitin • SUMO • Nedd8 • ubiquitin-like protein • functional proteomics • suicide inhibitor • protease
profiling • ubiquitin-specific protease • activity-based inhibitor • intein
Introduction
With the completion of genome sequences of many organisms, the assignment of biological function to newly discovered
gene products remains a challenge that cannot be addressed
by bio-informatics alone. Novel proteomics approaches are
crucial for the elucidation of protein function. Analysis of
expression levels using RNA and protein micro-arrays provides
at best indirect clues to the function of genes,1,2 but is not
necessarily a reliable predictor of protein activity levels within
the cell. Function-dependent methodologies are therefore
required to assess biological activity profiles of protein and
enzyme classes. Some chemistry-based efforts have been
directed toward different families of proteolytic enzymes.3,4
Small molecule libraries5 and mechanism-based molecular
probes that target active site residues of proteases in a covalent
fashion have been successfully applied to the serine,6 cysteine,7
and threonine protease families.8 Such probes are generally
based on a peptide scaffold that determines the specificity of
recognition, and a reactive group that traps the active site
nucleophile (reviewed in ref 4). Most of these reactive groups
are electrophilic covalent active-site modifiers, such as aldehydes, boronic acids, epoxides,7 vinyl sulfones,9 alkyl halides,
* To whom correspondence should be addressed. Huib Ovaa, Department
of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, room 836D,
Boston MA, 02115, USA. Phone: (617) 432-4779. Fax: (617) 432-4775.
E-mail: huib_ovaa@hms.harvard.edu
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Journal of Proteome Research 2004, 3, 268-276
Published on Web 02/06/2004
and fluorophospates.3 For proteomics purposes, an enzyme
should bind the inhibitor with sufficient affinity to survive
rigorous purification. The inhibitor, in turn, must contain a tag
for the retrieval and/or visualization of proteolytic enzymes
targeted. When applied to complex mixtures or cell lysates,
these inhibitors provide activity data on multiple members of
an enzyme class simultaneously, without the need for biochemical purification.
To date, a number of studies have utilized chemistry-based
approaches to identify and characterize enzymes involved in
specific biological processes. Epoxide-based probes have been
used to characterize the activity of cathepsins (a class of
cysteine proteases) during the uptake and degradation of
antigens for MHC II restricted antigen presentation.10 Small
libraries of probes that target cathepsin-like proteases of the
malaria parasite Plasmodium falciparum were used to define
the activity of proteases during the lifecycle of the parasite, and
to identify a specific inhibitor that blocks red blood cell
invasion.11 Peptide vinyl sulfone-containing probes, initially
developed to target cysteine proteases, were shown to react
also with the proteasome, a multicatalytic 2.4 mega-Dalton
threonine protease.8 These probes were then used to characterize antigen-processing pathways in the context of class I MHCrestricted antigen presentation.12 Fluoromethyl ketone containing molecules have been used to study the activity of
caspases in apoptosis;13 likewise, fluorophoshate derivatives can
10.1021/pr0341080 CCC: $27.50
 2004 American Chemical Society
Ub/UBL-Based Proteomics
Table 1. Ubiquitin-like Proteins to Which Intein-Based
Approach was Applieda
a Amino acid sequences used are derived from mouse clones described
in reference 36. Percentages identity apply to the processed forms of the
Ub-like proteins. The dash in the C-terminal sequence indicates the position
where processing occurs to generate the mature UBL protein. URM1, FAT10,
and Apg12 are expressed in their mature form. The C-terminal sequence of
Fau extends beyond the point shown. N.D.: not done.
target serine hydrolases, a large proteolytic enzyme family
involved in signaling and many other cellular processes.6
Further improvements on the current generation of reagents
will create probes with greater specificity and sensitivity for a
given family of enzymes and minimize cross-reactivity and
nonspecific labeling. This should allow new applications of the
reagents, such as in vivo activity measurements using fluorescent probes.4 To date, few of the available probes are cellpermeable, due to their size or charge properties, necessitating
cell lysis prior to reaction with the inhibitor. More recent
developments includes the synthesis of cell-permeable inhibitors that label distinct enzymes in living cells.14 These reagents
allow measurement of activity and retrieval of the relevant
enzymes under more physiological conditions.
In this review, we describe the development and generation
of a novel set of molecular probes that target proteases specific
for ubiquitin (Ub) and ubiquitin-like (UBL) proteins. These
probes are based on the full-length sequences of the respective
Ub or UBL proteins. Novel synthetic approaches were therefore
required for their synthesis. Activity profiles of entire enzyme
families active in crude extracts can be obtained with these
tools in a single experiment. In addition, these probes can be
used to retrieve and identify new members of the Ub and UBL
specific protease families.
Conjugation of Ubiquitin and Ubiquitin-like Proteins.
Ubiquitin and ubiquitin-like proteins are small proteins that
are conjugated post-translationally onto appropriate substrates,
usually proteins, thereby regulating a wide variety of cellular
processes. (Table 1).15,16 Many UBLs are related in both
sequence and 3-dimensional structure to Ub.17-22 The Cterminal residue of Ub or a UBL is conjugated to the -amino
group of a lysine residue in the target protein, to yield an
isopeptide bond; rarely, the N-terminal amino group of the
substrate is used for conjugation (Scheme 1). One UBL, Apg8,
and presumably its homologues GATE-16, MAP1-LC3, and
GABARAP is unique, in that it is conjugated to a membrane
lipid instead of another protein.23 A Ub/UBL-specific E1
activating enzyme or enzyme complex initiates the conjugation
cascade. E1 activates the C-terminus of Ub/UBL, via ATPdependent adenylate formation. This is followed by an intraenzymatic nucleophilic displacement, resulting in a thioester,
condensing an Ub/UBL molecule with the E1 active-site
cysteine. The activated Ub/UBL is then transferred to the active
site cysteine residue of an E2 conjugating enzyme. Multiple E2
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Scheme 1. Modification of Protein Substrates by Ub/UBLsa
a Ubiquitin (Ub) and ubiquitin-like molecules (UBLs) are expressed as precursors, either in form of concatemers or Cterminal extensions. A subset of Ub/UBL-specific proteases
cleaves precursor polypeptides to obtain the free Ub/UBL monomers (recognition and proteolysis of a peptide bond). Conjugation of Ub and UBLs to target polypeptides is achieved by several
enzymatic steps via a cascade involving E1, E2, and E3 enzymes
(see text). Conjugated protein substrates are then subjected to
deconjugation by Ub/UBL-specific proteases that specifically
recognize and cleave isopeptide bonds. Individual proteases may
recognize a specific subset of conjugated substrates.
Scheme 2. Removal of Ub/UBLs from Protein Substrates by
Cleavage of the Isopeptide Bonda
a Protein substrates conjugated with Ub/UBL are subjected to
proteolysis by a set of specific proteases. The catalytic center
harbors a nucleophilic cysteine residue (as well as an asparagineand a basic histidine residue). Attack of the N()-amide bond
followed by hydrolysis requires specific recognition of the
C-terminal part of Ub/UBL by the protease. Many of these
proteases may also recognize domains of the protein substrate
in order to induce efficient proteolysis.
enzymes are known for Ub, whereas single E2s seem to act on
other UBLs. Finally, Ub/UBL molecules are conjugated to their
substrates; in many cases, an additional player, an E3 ligase,
confers substrate specificity.15,16 While HECT-type E3 ligases
participate catalytically in the transfer of Ub to the substrate,
RING family E3s act as adaptor molecules, bridging the E2Ub complex together with the target protein.
Reversal of Ub/UBL Modification. The steady state level of
Ub/UBL conjugates is the result of a balance between the
action of ubiquitin ligases and Ub/UBL deconjugating enzymes.
Deconjugation is effectuated by specific proteases (Scheme 2).
Whereas each Ub/UBL molecule is apparently served by a
single E1-(like) activity, multiple Ub/UBL specific proteases
exist for most Ub/UBL modifiers.24,25 These proteases belong
to different families and play an important regulatory role by
Journal of Proteome Research • Vol. 3, No. 2, 2004 269
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determining the conjugation status of the substrate proteins.
Ub/UBLs are expressed as pro-protein precursors or in the case
of Ub as fusion proteins, requiring the action of Ub/UBLspecific proteases to generate the mature Ub/UBL molecules.16
Inspection of the genome sequences of human and mouse
suggests the presence of some 60-70 genes that encode
potential Ub/UBL deconjugating enzymes. The multiplicity of
these genes immediately raises the question of their biological
role. For a few of these enzymes, possible functions have been
identified. Furthermore, catalytic activity has been confirmed
for a limited number of these genes.26 Therefore, generation
of tools that allow for enzyme identification, and confirmation
of catalytic activity should greatly facilitate the study of these
putative enzymes.
Four major subfamilies of ubiquitin specific proteases (USPs)
have been identified to date. The best-studied subfamilies,
characterized by the presence of a catalytically active cysteine
residue, are known as ubiquitin-specific processing proteases
(UBP) and ubiquitin carboxy-terminal hydrolases (UCH). Members of these families possess the signature sequence motifs
of a cysteine protease and show characteristic patterns of
sequence conservation in their predicted core catalytic domains.26 UBPs can remove Ub from large polypeptides and
disassemble poly-Ub chains. UCHs are more active against Ub
with small C-terminal extensions, and can co-translationally
process Ub pro-proteins. They are, however, poor at cleaving
Ub-protein conjugates.27 We recently identified a novel USP
containing an ovarian tumor (OTU) domain, using a proteomics approach.28 This enzyme belongs to an distinct cysteine
protease family,29 with no similarity to UBPs and UCHs. Other
active members of this enzyme family were subsequently
shown to cleave poly-Ub conjugates, confirming the specificity
of OTU-domain containing proteases for Ub.30,31 Finally, a
JAMM family protease within the 19S cap of the proteasome,
RPN11 (POH-1), was shown to cleave ubiquitin from substrates
in a Zn2+- and ATP-dependent manner. RPN11 lacks a cysteine
protease signature, and is insensitive to the classical USP
inhibitor Ub aldehyde.32 Another JAMM family protease, Jab1/
Csn5, was shown to have deneddylating activity, removing
Nedd8 from cul1 thereby modulating SCF Ub ligase activity.33
Substrate Specificity of USPs and UBL-Specific Proteases.
In addition to their ability to hydrolyze Ub conjugates, some
members of the UCH and UBP families use UBL proteins as
their substrates. UCH-L3, UCH-L1, and USP5 (Iso-T), all have
activity against Nedd8, a UBL with the greatest homology to
Ub.33-36 Ubp43 (USP18), originally identified as a Ub-specific
protease, was subsequently shown to be specific for ISG15 and
may not act on Ub at all.37 SUMO deconjugation is performed
by cysteine proteases belonging to the ULP family.38,39 The ULP
proteases are unrelated in sequence to the UCHs and UBPs,
but structural data show that the configuration of the active
site residues and the catalytic mechanism are conserved
between these three cysteine protease families.40-42 One member of the ULP family, SENP8/DEN1, was shown to be specific
for Nedd8, not SUMO, as originally predicted based on
sequence comparison.36,43-45 Additionally, highlighting the
range of substrates acted upon by ULP family members,
adenoviruses encode a proteinase similar to ULPs with specificity for ubiquitin.46 For yeast Apg8 and its mammalian
homologues, a family of Apg4 cysteine proteases has been
identified, which has no resemblance to the other cysteine
protease families.47-49 It is clear that Ub/UBL processing and
deconjugation reactions are performed by a diverse set of
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Hemelaar et al.
proteases, with a particular UBL being served by members of
multiple protease families. Therefore, the assumption that Ub/
UBL-specificity of proteases can be inferred from the substrate
specificity of related enzymes is no longer valid. Instead, Ub/
UBL specificity of putative proteases needs to be determined
anew for each individual case. Additionally, more unidentified
Ub/UBL-specific protease families are likely to exist.
We have succeeded in targeting USPs and UBL-specific
proteases using a novel chemistry-based functional proteomics
approach, described below.28 Determination of enzymatic
activity for a given USP is usually made by measuring hydrolysis
of Ub-fusion proteins or of the fluorogenic substrate Ubaminomethylcoumarin (Ub-AMC50). These assays can be performed only on purified enzymes and do not allow the
measurement of activity of individual proteins in a crude lysate,
where associated factors may drastically alter the activity and
specificity of an enzyme.51-53 Because of differing enzymatic
reactivity toward Ub-AMC,50 it is not clear whether the use of
this model substrate would necessarily allow identification of
all members of the USP family. USPs may possess binding
specificity for the type of Ub-linkage used for conjugation as
well as the number of Ub moieties bound and sequences in
the target protein surrounding the site of Ub attachment. To
be able to dissect the specificity of USPs in complex systems,
novel tools, and synthesis strategies were clearly required. We
chose to develop suicide-substrate probes utilizing the intact
sequence of Ub to address these questions.
Design of Protein-Based Active Site-Directed Probes. Selection of an appropriate specificity element to target a particular
protease class ensures the labeling of the desired enzymes and
minimizes nonspecific modification. All USPs (with one exception) are cysteine proteases that cleave after the C-terminal GlyGly motif of Ub, (Table 1).26 Studies of reversible inhibitors and
substrates of USPs have indicated that the enzymes interact
with large areas of Ub in addition to recognizing the cleavage
site. Recent crystallographic analysis of the catalytic domain
of USP7 in complex with the USP inhibitor Ub-aldehyde (Ubal)
has revealed the structural details of UBP-Ub interactions.
Major structural changes take place in USP7 upon binding Ub,
bringing catalytic residues together to form a transition-state
intermediate ready for nucleophilic cysteine-mediated cleavage.42 These observations suggested that a large portion of the
Ub sequence would be required to achieve desired specificity
for USPs. Initially, we explored the use of peptide-based probes
corresponding to the C-terminus of Ub. The use of 8, 10, or
14-mer sequences did allow modification of purified USPs
(Overkleeft, Borodovsky and Ploegh, unpublished observations),
but the efficiency of labeling in crude lysates was much lower
than that observed for full-length Ub probes. We therefore
chose the entire sequence of Ub as a specificity element for
the development of covalent probes.
C-terminally modified Ub has been used previously to target
USPs. Ub-aldehyde and Ub-nitrile are potent and specific
inhibitors of USPs and have been valuable tools in the study
of this enzyme family.54,55 Complexes of proteases with Ubaldehyde or SUMO-aldehyde have been elegantly used in
crystallographic studies.41,42 However, the modification of proteases by these inhibitors is not generally compatible with
denaturing SDS-PAGE conditions. The success of Ubal and Ubnitrile in targeting USPs indicates that the active-site nucleophiles can be trapped if an appropriate electrophilic group is
placed at the C-terminus of Ub. Ub-aldehyde was generated
after borohydrate reduction of a Ub-UCH thioester. The
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Ub/UBL-Based Proteomics
Scheme 3. Synthesis of HAUb-Derived
Probesa
a The intein-based chemical ligation method. Recombinant
HAUb75-intein-chitin binding domain (CBD) fusion protein was
bound to a chitin affinity column; on-column cleavage of the
HAUb-intein junction was induced by the addition of β-mercaptoethane sulfonic acid (MESNa). The resulting HAUb75-MESNa
thioester was subjected to a chemical ligation, generating the
desired HAUb-derived probe. HA-tagged UBL-derived probes
were generated using the same strategy.
resulting Ub-UCH mixed hemi thioacetal is so stable that it
withstands further borohydride reduction. In uncomplexed
form Ubal is reduced by borohydride within seconds.
Vinyl sulfones are mechanism-based cysteine protease inhibitors that form stable covalent complexes with target
enzymes.9 Therefore, we synthesized an Ub-based probe that
harbors a glycine-vinyl sulfone moiety. Ub vinyl sulfone (UbVS)
was first prepared using a trypsin-mediated transpeptidation,
followed by chemical modification of the C-terminus of Ub.51
UbVS was designed so that the electrophilic carbon of the vinyl
sulfone is positioned in the same place as the Ub-terminal
carbonyl moiety in the natural substrate, thus placing it in the
optimal position for nucleophilic attack by the active site
cysteine of a USP. Nucleophilic Michael addition of an active
site cysteine residue onto the vinyl sulfone, results in the
formation of a covalent and robust thioether adduct. Indeed,
UbVS was demonstrated to covalently target a set of USPs in
cell lysates.51
Despite the success of UbVS in targeting USPs, the use of
[125I] for detection and the low synthetic yield limits its
usefulness. The design of epitope-tagged probes for the identification of the target-proteases, nonradioactive detection and
the extension of the approach to UBL sequences were desirable
extensions of this line of work. While carboxypeptidase Y
catalyzed transpeptidation can be used to modify the Cterminus of Ub, in our hands, the carboxypeptidase-based
protocols yield variable results. As with trypsin-catalyzed
transpeptidation, carboxypeptidase-based protocols are highly
dependent on sequence content and are not likely to be
generally applicable.
Synthesis of Ub/UBL-Based Active Site-Directed Proteomics Probes using an Intein-Based Chemical Ligation Method.
An alternative synthetic route was therefore needed to introduce N-terminal epitope tags and to generate C-terminal
electrophilic derivatives of UBL molecules (Scheme 3). We took
advantage of intein-based cloning and expression vectors. The
intein-based method relies on bacterial expression of a fusion
protein consisting of Ub/UBL-domain fused with an intein
domain and a chitin-binding domain (CBD) 56. The chitinbinding domain enables high affinity binding of the fusion
protein to a chitin column, allowing the isolation of the desired
protein in good purity using a one-step purification method.
The intein domain is a modified version of the intein from the
self-splicing protein VMA1 of Saccharomyces cerevisiae. The
desired Ub or UBL sequence is cloned upstream of the intein
domain with the omission of the C-terminal residue of the
processed form of Ub/UBL (a glycine residue, with the exception of HUB1 (Table 1, and refs 28,36,48). The intein undergoes
a reversible, intramolecular trans-thioesterification (N-S acyl
shift), which results in a thioester bond between the Ub/UBL
C-terminus and the intein. The equilibrium of the N-S shift
favors the peptide bond and therefore the fusion protein is
quite stable. In the presence of excess nucleophile, the thioester
is cleaved and the equilibrium is shifted to complete conversion, releasing a cleaved product from the resin (ref 56 and
references therein). Incubation of fusion proteins immobilized
onto the chitin column with excess thiol thus leads to the
release of the Ub/UBL as a thioester, as readily achieved by
the use of β-mercapto ethane sulfonic acid sodium salt (MESNa). Its overall negative charge diminishes hydrolysis rates,
but also makes the resulting proteins less susceptible to
nucleophilic displacement. The purified Ub/UBL-MESNa
derivative is then reacted with an electrophilic amine, generating the desired Ub/UBL-based probes. We have applied this
approach to Ub and the UBL thioesters listed in Table 1.
Versatility of the Intein-Based Chemical Ligation Method.
The intein-based strategy is compatible with several important
modifications, which greatly extend its scope. Using this
method, we generated functional probes for Ub, Nedd8, ISG15, SUMO-1, GATE-16, MAP1-LC3, GABARAP, and Apg8 with
or without epitope tags (Table 1),28,36,48 all of which were
expressed at acceptable levels. Some UBLs were unattainable,
due to low expression, or to insolubility of fusion proteins,
instability of intermediate MESNa thioester derivatives (i.e.,
hydrolysis), or intramolecular cyclization reactions. Increased
salt concentration and nonionic detergents are compatible with
the intein-CBD fusion based purification and cleavage procedure, and may allow increased recovery of intein-fusion
proteins. Thioester stability is hard to predict and is dependent
on nucleophilicity and flexibility of remote residues in the Ub/
UBL molecule.
The intein-based chemical ligation approach also lends itself
to the introduction of a diverse array of chemical functionalities
and can be used to synthesize enzyme substrates such as UBLAMC44 in addition to the inhibitors described here. Different
Michael acceptors as well as alkylhalide derivatives were
introduced at C-terminus of Ub (Figure 1A and ref 28). It was
demonstrated that different electrophilic groups conferred
differential reactivity toward USPs in cell lysates. The alkylhalide-containing probes modify a distinct set of polypeptides,
when compared to Michael acceptors. One alkylhalide derivative (HAUbBr2), a bromoethylamide probe, labels a unique
polypeptide not targeted by other Ub-based probes (Figure 1B
and ref 28). We identified this unique enzyme to be Hspc263,
an OTU domain containing protease subsequently termed
otubain1.28,29 The variation in labeling observed when a range
of C-terminal electrophilic traps is used demonstrates that USPs
do not possess identical active-site chemistry which most likely
reflects differences in their substrate specificity. Fine-tuning
of the target specificity of these probes may ultimately lead to
Journal of Proteome Research • Vol. 3, No. 2, 2004 271
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Hemelaar et al.
Figure 1. HAUb-derived active site-directed probes containing different C-terminal thiol-reactive groups show distinct labeling
specificities. A. List of C-terminal electrophilic groups. B. EL4 cell lysates (20 µg) were incubated with 0.5 µM of HAUb HAUbVS,
HAUbVME, HAUbVSPh, HAUbBr2, or 1 µM of HAUbCN, HAUbCl, HAUbBr3 as indicated. The labeled proteins were resolved by 8%
reducing SDS-PAGE and immunoblotted with anti-HA antibody. Polypeptides referred to in the text are indicated by arrowheads.28
the development of reagents capable of modifying individual
proteases. Since the Ub/UBL processing pathways have been
implicated in human disease, development of drugs targeting
individual proteases may be of pharmaceutical interest.
Since the intein-based fusion proteins are expressed recombinantly in E. coli, epitope tags can easily be incorporated into
the proteins. The availability of high-affinity antibodies enables
nonradioactive detection of the epitope-tagged probes and
adducts as well as their immunopurification. For most probes,
the inclusion of an N-terminal influenza hemagglutinin (HA-)
epitope tag did not interfere with protein expression and
purification. Labeling efficiency and selectivity of the resulting
probes were likewise unaffected when compared to [125I]labeled, untagged probes.28,36,48,51 Incorporation of the HA-tag
into the Nedd8 sequence resulted in the insolubility of the
MESNa product, a problem overcome by the use of a FLAG
epitope tag.36,44,51 Production of fusion proteins in bacteria
further enables the introduction of point mutations. The
presence of cysteine residues may be incompatible with the
intein-based since they can react with the C-terminal electrophile. This may be avoided by prior mutation.
Application of Ub/UBL Derived Active Site-Directed
Probes: Chemistry-Based Functional Proteomics. N-terminally epitope tagged probes can be used similarly to the radioiodinated versions to detect the modified enzymes after the
separation of the crude protein mixture by gel electrophoresis.28,36,48,57 This methodology can be used to visualize multiple
active enzymes in cell lysates simultaneously, i.e., profiling of
active proteases. In yeast, deletion mutants of predicted USPs
led to the identification of the enzymes targeted by [125I]UbVS.51 Since a genetic approach is not straightforward in
mammalian cells, and requires prior knowledge of potential
targets, USP identification and profiling was achieved using a
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Journal of Proteome Research • Vol. 3, No. 2, 2004
functional proteomics approach. This method entails the
incubation of epitope tagged Ub/UBL probes with cell lysate,
followed by the immunopurification of the covalently bound
adducts using antibody-Sepharose beads. The retrieved proteins are separated by SDS-PAGE and visualized by Coomassie
or silver staining (Figure 2). Relevant sections of gels are then
excised, subjected to in-gel trypsinolysis and analyzed by
tandem mass spectrometry. Immunopurification can be performed either under native or denaturing conditions, which
allows discrimination of enzymes covalently modified by the
active site-directed probes from proteins that merely interact
in a noncovalent fashion (Figure 2 and refs 28,57). This
approach led to the identification of 23 active USPs in mouse
thymoma cells and 11 active USPs in human B-cell tumors. In
addition, proteases specific for Nedd8, ISG15, GATE-16, MAP1LC3, GABARAP, and Apg8L were also shown to react with UBLbased probes.36,48 These identifications relied on the reactivity
of the proteases toward the Ub/UBL C-termini, directly demonstrating their activity. Nevertheless, the identification of these
proteases was not biased by assumptions as to specificity for
a particular UBL.
Active Site-Directed Probes Reveal Novel Members of the
USP Family. Most of the 23 proteases with specificity for Ub,
identified in the EL4 mouse thymoma cells line,28 belong to
the UCH and UBP families of Ub specific proteases, although
for several of these proteins activity had not been demonstrated
previously. Among the labeled USP species was USP14, which
appears to be active only when bound to the 26S proteasome
complex, an observation that was also made for its yeast
homologue, Ubp6.51,52 Another Ubp identified was CYLD, a
protein encoded by a tumor suppressor gene mutated in
hereditary cylindromatosis.58 Our study was the first demonstration of USP-like activity of CYLD, a protein with an active
Ub/UBL-Based Proteomics
reviews
Figure 3. Ub-specific proteases are expressed and active in a
cell-type dependent manner.USP activity profiling of human
cancer cell lines. Cell lysates were incubated with HAUbVME,
separated by 8% SDS-PAGE, and immunoblotted with the antiHA monoclonal antibody 12CA5. Distinct activity profiles are
obtained for cancer cell lines. UCH-L1, an abundant USP in the
brain (and therefore the astrocytoma cell line) was shown to be
highly active in the B-cell malignancies multiple myeloma and
non-Hodgkin’s lymphoma.
Figure 2. Protein complexes containing HAUbVS modified
enzymes can be recovered by anti-HA immunoprecipitation.Silver
stain of HAUbVS modified proteins under different conditions.
5 mg of lysate were treated with 6.6 µg of HAUbVS or HAUb, a
3-fold molar excess of untagged UbVS was added to the
indicated sample. “Denatured” samples were treated with 0.4%
SDS prior to the addition of anti-HA agarose. Bound proteins
were eluted with 50 mM Glycine pH 2.5, resolved by 8% reducing
SDS-PAGE and silver stained.28
site signature divergent from that of most other UBPs. CYLD
was recently shown to negatively regulate NF-κB by cleaving
the K63 linked Ub chain from TRAF2, a function that may
contribute to the pathogenesis of cylindromatosis.58-62 The
functional proteomics approach also demonstrated that an
ovarian tumor (OTU) domain containing protein (HSCP263,
otubain 1) is a Ub specific protease.28,29 Several subsequent
studies confirmed that OTU-containing proteases, referred to
as otubains, indeed have the ability to bind Ub, and to remove
Ub moieties from substrates.28-31 The OTU family contains
numerous proteins that share the predicted protease signature;
the substrate specificity of these putative proteases remains a
fascinating question.
USPs in Malignant and Virus Infected Human B Cells. Ubderived probes were also used to study the activity of Ubspecific proteases in B cell malignancies (Figure 3 and ref 57).
In Burkitt Lymphoma derived cells, increased USP activity was
detected as compared to EBV transformed lymphoblastoid cells
(LCLs). Thus, increased USP activity correlated with their
elevated tumorigenicity.63 Eleven active USPs were identified
in these B-cell tumors. USP activity levels in Burkitt’s lymphoma and multiple myeloma cell-lines were measured and
Figure 4. Ub/UBL-specificity of protease families. Each arrow
indicates that at least one member of the protease family is
specific for the Ub/UBL to which the arrow points. Red arrows
represent protease family-Ub/UBL combinations discovered and/
or confirmed by the functional proteomics approach using the
Ub/UBL probes generated by the intein-based method. Black
arrows indicate protease family-Ub/UBL combinations determined by other methods. The proteases represented by the
numbered arrows are as follows (references in brackets): 1.
HSPC263, otubain 1 and 2 and Cezanne;28,30,31 2. UCH-L1, UCHL3, and UCH37 (ref 28 and references therein); 3. UCH-L1 and
UCH-L3;34,36 4. 20 members of the UBP/USP family were identified/confirmed as Ub-specific proteases using Ub-derived probes
(ref 28 and references therein). 5. USP21;35 6. UBP43 and IsoT/
USP5;36,37 7. Rpn11;32 8. Jab1/Csn5;33 9. SENP8/DEN1/NEDP1;36,43-45
10. Ulp1 and 2 (yeast), SENP1/SuPr-2, SENP2/SuPr-1/Axam,
SMT3IP2/Axam2, SUSP1, SMT3IP1;38,39,66,67 11. Apg4 (yeast),
Apg4A, Apg4B.47-49,68
compared to LCLs and primary B- and T-cells, leading to the
observation that levels of active enzyme UCH-L1 (PGP9.5), a
USP, are upregulated only in malignant B-cells and not in
healthy primary tissue or EBV-immortalized LCLs. Furthermore,
Journal of Proteome Research • Vol. 3, No. 2, 2004 273
reviews
the activity of several USPs in primary B and T cells, including
USP 5, 7, 9, and 15, changes dramatically upon stimulation with
various mitogens. It appears that these activities are generally
increased upon cell proliferation induced either by mitogen
or virus-dependent activation. In contrast, up-regulated UCHL1 activity is observed only in a select range of tumors,
suggesting that UCH-L1 may contribute to increased tumorigenicity and decreased immunogenicity of these tumors.
Several Proteases Exhibit Specificity for Ub and UBL
Proteins. Using radio-iodinated probes, we detected six putative proteases for Nedd8, three for ISG15 and four for SUMO-1
(Figure 4 and ref 36). N-terminally epitope tagged versions of
the respective probes were employed to identify several of these
proteases. UCH-L1, UCH-L3, and SENP8 (DEN1) were identified as Nedd8-specific proteases. Dual specificity for Ub and
Nedd8 has been described for UCH-L3, but this was the first
time it was demonstrated for UCH-L1.34 Dual specificity for Ub
and Nedd8 may be a general property of proteases of the UCH
family, as it has also been described for the yeast UCH enzyme
Yuh1.40,64 SENP8 shares homology with members of the ULP
family of SUMO-specific proteases.39 Surprisingly, SENP8 was
found to react with FLAG-Nedd8-VS and in another study, to
hydrolyze Nedd8-AMC, suggesting that Nedd8 is SENP8’s true
substrate.36,44 Furthermore, isopeptidase T/USP5 was identified
as an adduct with ISG15-VS. This indicates that IsoT possesses
a protease activity toward the ISG15 C-terminus in addition to
its previously established Ub-specific activity. We have proposed that IsoT may function as an ISG15 processing protease.36
An earlier study has reported that an activity the size of IsoT
can process ISG15/UCRP precursors, but the identity of this
enzyme has not been established.65 The functional proteomics
approach has therefore highlighted the fact that identification
by sequence alignment alone of the enzyme family to which a
UBL-specific protease belongs, does not necessarily predict its
substrate specificity.
Vinyl sulfone derivatives of the mammalian Apg8-like proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L were instrumental in the identification of a single protease, Apg4B, as the
enzyme specific for the C-termini of these highly divergent
Apg8-like proteins.48 Apg4B was unequivocally identified by
tandem mass spectrometry as an adduct with HA-MAP1-LC3VS, directly excluding other members of the mammalian Apg4
family. The Apg4B protease complements the Apg7 activating
and Apg3 conjugating enzymes necessary for post-translational
modification of the Apg8-like proteins, and the identification
of Apg4B will likely aid in the further elucidation of the role(s)
of the Apg8 homologues in the cell.
Future Perspectives. Functional proteomics is not the only
application of C-terminally modified Ub/UBL probes. Such
mechanism-based active site-directed inhibitors can be used
to assess the activity of multiple UBL-specific proteases in
parallel. Presence and activity levels of individual proteases can
be compared between samples; for example, one may compare
different cell lines, or tissues, representing health and disease
states, after drug treatment, or following viral infection. These
probes have an advantage over antibodies in that they can
detect multiple enzymes in parallel and in that they detect
activity levels rather than protein levels. Comparative studies
addressing expression levels and activity might provide novel
information about regulation of protein function, especially at
the posttranslational level. The case of USP14 and its yeast
homologue has shown the importance of this distinction.51,52
The ability to equip Ub and UBLs with different types of
274
Journal of Proteome Research • Vol. 3, No. 2, 2004
Hemelaar et al.
electrophilic groups may allow the targeting and identification
of new classes of UBL-specific proteases, as we have observed
for Ub. Further exploration of different electrophilic substituents may also allow fine-tuning of the target specificity of the
probes and may lead to the development of inhibitors specific
for single proteases. In addition, many USPs may have increased affinities for poly-ubiquitin structures. It will therefore
be desirable to generate probes that contain multiple Ub
moieties. A combination of enzymatic and chemical approaches will be required to achieve this goal.
The intein-based method combined with chemical ligation
should allow the preparation of branched ubiquitin molecules
that include lysine-containing peptides attached to the Cterminus of Ub/UBL via an isopeptide bond. Such reagents may
be useful to assess substrate specificity of USPs. On the basis
of the complexity of the USP gene family, each individual
enzyme may target a specific set of substrates, and their
identification may hold clues about the enzyme’s biological
function.
Although the probes described here react solely with Ub/
UBL-specific proteases in cell lysates, we have shown that they
can covalently react with purified E1 activating enzyme, E2
conjugating enzyme and a E3 HECT ligase in vitro 36. Whereas
the physiological thioester intermediates of UBL-enzyme
complexes are very labile, the thioether adducts generated with
the probes are stable and might be suitable for structural
analysis by X-ray crystallography. This could yield important
insights into the mechanisms of action of these enzymes.
Finally, it will be important to render the probes cellpermeable, which will allow us to study the effect of inhibition
of a specific set of UBL-proteases on cellular physiology.
Abbreviations
Ubiquitin (Ub); Ubiquitin-like protein (UBL); Ubiquitin
carboxy-terminal hydrolase (UCH); Ubiquitin processing protease (UBP); Ubiquitin specific protease (USP); Ovarian tumor
domain (OTU); Ubiquitin-like protease (ULP).
Acknowledgment. This work was supported by a VENI
grant from the Netherlands Organization for Scientific Research
(HO) and by grants awarded by the National Institutes of Health
(HLP), and by the Multiple Myeloma Research Foundation and
the Harvard Center for Neurodegeneration and Repair (BMK).
PJG was supported by a NIH training grant. Figures 1 and 2
were reprinted28 with permission from Elsevier.
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