O-GlcNAc peptide epoxyketones are recognized by mammalian proteasomes

Article abstract of DOI:10.1021/ja901231w

(Chemical Equation Presented) Cytosolic and nuclear proteins may be subject to both O-GlcNAcylation and proteasomal degradation. By means of activity-based profiling, we demonstrate that O-GlcNAc serinecontaining peptide epoxyketones bind to the proteasome catalytic active sites and thus provide the first clear evidence that proteasomes recognize peptides post-translationally modified with a GlcNAc moiety.

Full text of DOI:10.1021/ja901231w

Published on Web 08/06/2009
O-GlcNAc Peptide Epoxyketones Are Recognized by Mammalian
Proteasomes
Martin D. Witte, Bogdan I. Florea, Martijn Verdoes, Oloruntosin Adeyanju, Gijs A. Van der Marel, and
Herman S. Overkleeft*
Leiden Institute of Chemistry, Leiden UniVersity, P.O. Box 9502, 2300 RA Leiden, The Netherlands
Received February 17, 2009; E-mail: h.s.overkleeft@chem.leidenuniv.nl
Proteasomes are multicatalytic proteinase complexes responsible
for the degradation of cytosolic and nuclear proteins. In eukaryotes,
proteasome substrates are normally marked for degradation through
the attachment of ubiquitin chains at specific sites, and subsequent
proteasome-mediated degradation takes place in a controlled and
ATP-dependent fashion.1 In mammals, some oligopeptides gener-
ated by proteasomes are presented to the immune surveillance
system by major histocompatibility complex class I (MHC I)
molecules.2 Next to self-peptides, peptides derived from virally
encoded proteins are also exposed at the cell surface in this fashion,
and thus, mammalian proteasomes partake in combating viral
infections. Mammals express several proteasome particles that
appear to play specific roles in MHC I-mediated immune response.
These are the constitutively expressed 26S proteasome, the immu-
noproteasome, and the recently discovered thymoproteasome. Of
these, the latter two are expressed by the specific tissues where
they exist next to the 26S proteasome.3 In all mammalian
proteasomes, the actual proteolytic activities reside in the core 20S
particles, which are C2-symmetric barrels composed of two rings
Figure 1. (A) Schematic representation of the experiment. (B) Structure
of GlcNAcylated proteasome probes 1-4 and control probe 5. Az )
Azidoacetyl, EK ) Epoxyketone.
of seven distinct ꢀ-subunits (ꢀ1-ꢀ7) sandwiched between two rings
composed of seven distinct R-subunits (R1-R7).4 In 26S protea-
somes, ꢀ1, ꢀ2, and ꢀ5 are catalytically active and cleave prefer-
entially after acidic, basic, and hydrophobic residues, respectively.5
These subunits are replaced by ꢀ1i, ꢀ2i, and ꢀ5i in immunopro-
teasomes and by ꢀ1i, ꢀ2i, and ꢀ5t in the thymoproteasome. The
exact role and substrate preference of the combined seven catalytic
subunits is the subject of extensive studies, and the same holds
true for elucidating the nature of the peptides produced by
proteasomes and recruited by the immune system. In this context,
the immunogenicity of MHC I-associated phosphopeptides is well-
established, and this implies the involvement of proteasomes in
the turnover of phosphorylated proteins.6
In the past decades, another major post-translational modification
that occurs in the cytoplasm and the nucleus, namely, protein Ser/
Thr O-GlcNAcylation, has drawn considerable attention.7 O-
GlcNAcylation is a reversible process, in that O-GlcNAc transferase
(OGT) glycosylates a serine or threonine residue and a hexosamini-
dase (O-GlcNAcase) is responsible for the removal of O-GlcNAc
residues. Protein O-GlcNAcylation and proteasomal degradation
occur in the same cellular compartments, and it is therefore not
surprising that the two processes interface. O-GlcNAcylation of
the proteasome down-regulates its activity, whereas O-GlcNAcy-
lation of certain proteins appears to give some protection against
proteasomal degradation.8 At the same time, O-GlcNAcylated
MHC I-derived peptides were evidenced in several studies, indicat-
ing that O-GlcNAcylated proteins can act as viable proteasome
substrates.9 In regard to whether the proteasome is capable of
degrading O-GlcNAcylated proteins, we felt that activity-based
profiling using tailored O-GlcNAcylated proteasome inhibitors
would give insight into this uncharted matter in a straightforward
fashion. To this end, we prepared (Figure 1) a set of four peptide
epoxyketones based on the selective proteasome inhibitor epoxo-
micin10 and extended with an N-terminal GlcNAcSer derivative.
In compounds 1 and 2, the N-acetyl group of glucosamine is
replaced by a N-(azidoacetyl) group, and the pentapeptide is
N-terminally acetylated. Compounds 3 and 4 differ in that the
azidoacetyl group is placed at the N-terminus of the peptide and
the glucosamine nitrogen is acetylated. Both peracetylated (1 and
3) and unprotected (2 and 4) derivatives were prepared following
the block-coupling strategy we routinely use for the construction
of C-terminally modified oligopeptide proteasome inhibitors.11
The ability of peptide epoxyketones 1-4 to inhibit the protea-
some was assessed indirectly in a competition experiment using
the pan-reactive and fluorescent proteasome activity-based probe
MV151 as a read-out.12 To this end, lysate derived from HEK cells
(a human hybridoma cell line expressing the constitutive protea-
some) was treated with increased peptide epoxyketone concentra-
tions prior to treatment with MV151, and the proteins were
separated by SDS-PAGE. The fluorescence read-out (Figure 2A,
panels 1-4) revealed that the four probes are inhibitors having
comparable potencies and show a slight preference for the ꢀ5
subunit with respect to the individual catalytic activities. In all
instances, labeling with MV151 is largely abolished at 10 µM final
concentration. In the key experiment, samples of cell lysate
exposed to 1-4 at the same final concentrations were treated with
biotinylated phosphine.11 In this way, proteasome subunits modified
with an azide are directly visualized by Staudinger-Bertozzi
bioorthogonal labeling13 followed by SDS-PAGE and Western
blotting. The resulting images (Figure 2B, panels 1-4) reveal a
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10.1021/ja901231w CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Competition and labeling studies of proteasome probes 1-5. (A) Lysate from HEK293T cells (10 µg of total protein) was incubated with
increased concentrations of 1-5 and epoxomicin as a control for 1 h at 37 °C. Residual proteasomal activity was labeled by incubation with MV151 (1 µM
final concentration) for 1 h at 37 °C. After denaturation and resolution by SDS-PAGE, the potency was determined using fluorescence scanning. (B) Streptavidin
blot of HEK293T lysate (10 µg of total protein) treated with increased concentrations of 1-4 for 1 h at 37 °C, after which the azide was modified by
incubation with the Staudinger-Bertozzi reagent (400 µM final concentration) for 1 h at 37 °C. (C) Labeling of the probe in living HEK293T cells. Some
1 × 106 HEK cells were incubated with the indicated amounts of probes 1 and 3 for 16 h, after which the cells were harvested, lysed, and treated with either
MV151 (upper panels) or the Staudinger-Bertozzi reagent (lower panels).
signal that is highly complementary to the one witnessed in
panels1-4 of Figure 2A. The disappearance of MV151 labeling is
counterbalanced by the appearance of the streptavidin-horseradish
peroxidase-mediated signal, and the latter can only be the result of
covalent azide introduction onto the proteasome active sites. This
is the expected result for compounds 3 and 4, which have the azide
in the peptide backbone (indeed, these results corroborate prior work
from our laboratory).14 Peptide epoxyketones 1 and 2, however,
give equally efficient Staudinger-Bertozzi labeling. We conclude
that hexosaminidase-mediated removal of the GlcNAz moiety is
not a prerequisite for proteasomal entry and active-site modification
by these activity-based probes. In fact, concomitant treatment of
lysate incubated with 1 or 2 with hexosaminidase inhibitor NAG-
thiazoline15 did not give a discernibly different outcome.11 Perhaps
surprisingly, proteasomes not only bind O-GlcNAcylated peptide
epoxyketones but do so with a potency only slightly lower than
that of the parent compound epoxomicin (Figure 2A, panel 6). To
establish whether the steric bulk represented by the GlcNAc
moieties in 1-4 causes this slight loss in inhibitory potential or if
specific structural features play a role, we prepared and assessed
tyrosine derivative 5. This compound actually proved the most
potent of the series (Figure 2A, panel 5), leading to the tentative
conclusion that GlcNAc moieties are accepted by the proteasome.
Blocking proteasome activity in live cells necessitated increased
concentrations of 1 or 3 but otherwise gave essentially the same
picture, demonstrating the use of our probes also in this physi-
ologically more relevant research setting (Figure 2C). Our results
thus strongly point toward a natural role for proteasomes in
producing Ser/Thr O-GlcNAcylated peptides, some of which may
end up on the cell surface as part of MHC I complexes. Whether
such complexes are immunologically relevant in health and disease
remains an open question. Future research in our laboratory is aimed
in this direction through the generation of Ser/Thr O-GlcNAcylated
peptide epoxyketones more closely resembling actual proteasome
products/MHC I peptides.
Acknowledgment. This work was supported by The Nether-
lands Organization for Scientific Research (NWO) and The
Netherlands Proteomics Centre (NPC).
Supporting Information Available: Synthesis and characterization
of compounds 1-5, biological assays, structures of MV151 and
biotinylated phosphine, and complete ref 12. This material is available
free of charge via the Internet at http://pubs.acs.org.
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