Affinity labeling of the proteasome by a belactosin A derived inhibitor

Article abstract of DOI:10.1016/j.bmcl.2008.08.073

Belactosin A is a potent proteasome inhibitor isolated from Streptomyces metabolites. Here we show that a hydrophobic belactosin A derivative, dansyl-KF33955, can covalently, and specifically, affinity label the catalytic subunits of the 26S proteasome, which consists of the 20S protein degrading core particle and the 19S regulatory particles. The labeling of catalytic subunits proceeds faster in intact proteasomes in vivo than in isolated 20S core particles. These data suggest that the 19S regulatory particle may facilitate entry of the inhibitor into the 20S core particle. This cell-permeable chemical probe is an excellent tool with which to study the interactions of this proteasome inhibitor with proteasomes in intact cells.

Full text of DOI:10.1016/j.bmcl.2008.08.073

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5668–5671
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Affinity labeling of the proteasome by a belactosin A derived inhibitor
a
a
a
Makoto Hasegawa a, , Kazuhiro Kinoshita , Chika Nishimura , Umechiyo Matsumura ,
*
Masashi Shionyu a, Shun-ichi Ikeda b, Tamio Mizukami a
a Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, Tamura-cho 1266, Nagahama, Shiga 526-0829, Japan
b Bio-Frontier Laboratories, Kyowa Hakko Kogyo Co., Asahi-cho 3-6-6, Machida, Tokyo 194-8533, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Belactosin A is a potent proteasome inhibitor isolated from Streptomyces metabolites. Here we show that
a hydrophobic belactosin A derivative, dansyl-KF33955, can covalently, and specifically, affinity label the
catalytic subunits of the 26S proteasome, which consists of the 20S protein degrading core particle and
the 19S regulatory particles. The labeling of catalytic subunits proceeds faster in intact proteasomes
in vivo than in isolated 20S core particles. These data suggest that the 19S regulatory particle may facil-
itate entry of the inhibitor into the 20S core particle. This cell-permeable chemical probe is an excellent
tool with which to study the interactions of this proteasome inhibitor with proteasomes in intact cells.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 30 April 2008
Revised 8 August 2008
Accepted 20 August 2008
Available online 26 August 2008
Keywords:
Affinity label
Anti-cancer drug
Protease inhibitor
Proteasome
The 26S proteasome is a large protein complex containing en-
zymes that degrade proteins conjugated to ubiquitin and plays a
central role in selective proteolysis in eukaryotes. The 26S protea-
some is composed of the 20S core particle (CP) bearing several cat-
alytic sites for protein degradation and two 19S regulatory
particles (RP) involved in the recognition and unfolding of ubiqui-
tinated proteins.1 Structurally, the 20S CP is a 700-kDa complex
with a hollow cylindrical-shape composed of four stacked hepta-
meric rings. The two inner rings each consist of seven b subunits
apy, as well as providing a tool for understanding the ubiquitin-
proteasome system.2 We previously identified the novel protea-
some inhibitor belactosin A (Fig. 1, Structure 1) from Streptomyces
metabolites and showed that this compound inhibits human can-
cer cell proliferation.3 Interestingly belactosin A is a strong inhibi-
tor of the chymotrypsin-like activity in the purified 20S
proteasome CP in vitro.3 Structure-function studies indicate that
the b-lactone ring of belactosin A is necessary for full inhibition
of proteasome activity.3 More recently, an X-ray crystallographic
analysis revealed that a belactosin analog, homobelactosin C, cova-
lently binds to the active sites of the b5 subunit through esterifica-
tion of the b-lactone ring to a threonine residue that is present in
each catalytic center.4 Thus, the fundamental mechanism by which
peptidase activities in CP are inhibited by belactosins has been
clarified. However, binding of the inhibitors to the 26S proteasome,
the intracellular complex of CP and RP, is not well understood. RP
may regulate inhibitor entry into the 20S CP cavity. To analyze
intracellular interactions between inhibitors and the intact 26S
proteasome, a cell-permeable affinity-labeling probe derived from
a potent proteasome inhibitor is needed.
A hydrophobic belactosin A derivative, KF33955 (Fig. 1, Struc-
ture 2), in which a benzyl group has been introduced at the car-
boxyl group of belactosin A, exhibits a 100-fold greater growth
inhibition of HeLa cells, relative to belactosin A, which is presum-
ably due to the improved cell-membrane permeability of
KF33955.3 To detect covalent complexes of the belactosin A deriv-
ative and proteasomal components with high sensitivity, we pre-
pared dansyl-KF33955 (Fig. 1, Structure 3) as an affinity probe. In
this report, we demonstrate the successful labeling of b subunits
and the two outer rings each consist of seven
a subunits, providing
an enclosed cavity in which target proteins are degraded. Openings
at the two ends of the CP enable proteins to enter the cavity. Each
end of the CP associates with an RP that contains ubiquitin binding
sites and ATPase active sites. Thus, the RP recognizes, unfolds and
translocates target proteins into the catalytic cavity for degrada-
tion. Within the catalytic cavity, three b subunits (b1, b2 and b5)
contain peptidase active sites for caspase-like peptidylglutamyl
protein hydrolyzing (PGPH), trypsin-like, and chymotrypsin-like
activities, respectively. Protein degradation by the proteasome
plays key regulatory roles in metabolic adaptation, cell differentia-
tion, cell cycling, stress response, regulation of transcriptional fac-
tors, as well as generation of epitopes presented by the major
histocompatibility complex class I receptors and quality control
through removal of abnormal proteins.
Small molecule inhibitors of the proteasome are important tools
in developing new molecular-targeting drug for cancer chemother-
* Corresponding author. Tel.: +81 749 64 8100; fax: +81 749 64 8138.
E-mail address: m_hasegawa@nagahama-i-bio.ac.jp (M. Hasegawa).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.073

M. Hasegawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5668–5671
5669
O
H
N
H2N
N
H
O
CO2H
O
O
O
H
N
H2N
N
H
O
O
O
O
O
O
O
H
N
H
N
S
N
N
H
H
O2
N
O
O
O
O
O
Figure 1. Structures of belactosin A (1), KF33955 (2) and dansyl-KF33955 (3).
responsible for peptidase activities and compare the temporal pat-
tern of labeling between purified CP in vitro and the 26S protea-
some in vivo.
Belactosin A was isolated from the broth of Streptomyces
KY11780 cultures, and KF33955 was prepared from belactosin A,
as described previously.3 Dansyl-KF33955 was prepared by cou-
in the amino terminus of belactosin A. However, it is of interest
that introduction of the dansyl moiety to KF33955 markedly in-
creased the inhibitory effect on the chymotrypsin-like site, which
is thought to be the major site of protein degradation by
proteasomes.6
Since belactosin A represses proliferation of cancer cells3, we
compared the ability of the belactosin A derivatives to inhibit HeLa
cell growth (Table 1).8 The hydrophobic derivative KF33955 exhib-
ited a more potent inhibition of cell proliferation compared to the
parent compound, belactosin A. This high potency may be the re-
sult of the increased membrane permeability of KF33955. The
inhibitory effect of dansyl-KF33955 on cell proliferation is greater
than belactosin A, but 20-fold less potent than KF33955, suggesting
that the dansyl moiety reduced the membrane permeability con-
comitantly with the enhanced potency of the proteasome inhibi-
tory activity. Given that addition of a biotin moiety to belactosin
A severely decreased its inhibitory potency in HeLa cells (IC50 value
pling
a
succinimidyl ester of dansylaminohexanoic acid to
shows the MSMS spectrum of dansyl-
KF33955.5 Figure
2
KF33955 (precursor ion m/z 806.37). The mass signals m/z
347.14, 389.20, 418.17, and 698.32 were consistent with the pre-
dicted sizes of its cleavage products (Fig. 2 inset). Most of other sig-
nals in the low mass range (indicated by the asterisks in Fig. 2)
were the same values as those observed in MSMS measurements
of KF33955 (data not shown). These data are in full agreement with
the structure proposed.
To examine inhibition by belactosin A derivatives and other
well-known proteasome inhibitors, such as clasto-lactacystin b-
lactone and MG132, different concentrations of each compound
were added to reaction mixtures containing purified human eryth-
rocyte 20S CP and a synthetic substrate specific for each pepti-
dase.6,7 The calculated IC50 values are shown in Table 1.
Dansylation of the hydrophobic belactosin A (KF33955) resulted
in a marked enhancement of the inhibition of the chymotrypsin-
like activity and PGPH activity. Dansylation reduced inhibition of
the trypsin-like activity, probably due to loss of the positive charge
of ꢀ350
lM, data not shown), dansylation appears to provide a
more advantageous affinity label for tracing labeled components
of the proteasome, as reported previously.9
To examine the binding characteristics of dansyl-KF33955 to
the proteasome in vivo, we first determined an inhibitor concen-
tration appropriate for clear detection of labeled catalytic b sub-
units by Western blotting using anti-dansyl antibody. When
HeLa cells were cultured in the presence of 1 lM inhibitor, labeled

5670
M. Hasegawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5668–5671
Intensity (x 107)
418.17
*389.20
2.50
2.25
2.00
1.75
1.50
418.18 389.20
O
O
H
N
H
N
N
S
N
H
N
H
O2
347.14
O
O
O
O
698.32
O
*218.12
1.25
1.00
0.75
0.50
0.25
0.00
343.20
480.23
786.38
508.22
*253.15
347.14
698.32
700
*299.21
300
*172.11
*291.13
150
200
250
350
400
450
500
550
600
650
750
800
850 m/z
Figure 2. The MSMS spectrum of dansyl-KF33955. The asterisks indicate common peaks of KF33955.
Table 1
dansyl-KF33955(1μM)
lactacystin
A
IC50 values of human erythrocyte 20S proteasome enzymatic activities and growth
inhibition of HeLa cells by inhibitors
Proteasome inhibitiona
(lM)
Growth
0.01 0.05 0.1 0.5
1
(μM)
25
Compound
inhibitionb
(lM)
β2
Chymotrypsin-
like activity
Trypsin- PGPH
like
(kDa)
activity
activity
β1
β5
Belactosin A
KF33955
Dansyl-KF33955
clasto-lactacystin
b-Lactone
0.82
0.34
0.029
0.029
4.9
0.45
2.1
14
3.7
0.15
8.3
37
0.50
9.8
ND
20
0.69
dansyl-KF33955(1μM)
B
MG132
0.011
2.1
0.12
ND
MG132
ND: not determined.
(μM)
0.01 0.05 0.1 0.5
1
a
IC50 values of human 20S proteasome activity.
IC50 values of growth in HeLa cell cultures.
b
β2
(kDa)
25
20
β1
β5
subunits could be detected without notable cell death (data not
shown). Figure 3 shows dansyl-KF33955 labeling of the catalytic
subunits in proteasomes in HeLa cells (in vivo) and its competitive
inhibition by various concentrations of clasto-lactacystin b-lactone
and MG132.10 The bands detected by the anti-dansyl antibody
were confirmed to be the b1, b2, and b5 subunits by performing
Western blotting using antibodies specific for each subunit (data
not shown). After incubation of HeLa cells with clasto-lactacystin
b-lactone, dansyl-KF33955 only labeled the b1 subunit suggesting
that dansyl-KF33955 and clasto-lactacystin b-lactone compete
with another for binding to the same sites in the b2 and b5
subunits (Fig. 3A). After incubation of HeLa cells with MG132, dan-
syl-KF33955 only labeled the b2 subunit, suggesting that dansyl-
KF33955 and MG132 compete with one another for binding to
the same sites in the b1 and b5 subunits (Fig. 3B). These results
indicate that dansyl-KF33955 specifically binds to the b1, b2 and
b5 subunits of 20S CP in vivo. Figure 4A shows the time-dependent
labeling of catalytic subunits in proteasomes in HeLa cells (in vivo)
Figure 3. Western blotting of dansyl-crosslinked subunits of the 26S proteasome
treated with 1 M dansyl-KF33955 in intact HeLa cells in the presence of vatious
concentrations of clasto-lactacystin b-lactone (A) and MG132 (B).
l
and Figure 4B shows the labeling in the 26S proteasome extracted
from HeLa cells (in vitro).11,12 Figure 4C shows labeling of subunits
in the similar condition except for using the purified human eryth-
rocyte 20S CP.12 Within as little as 0.5 h, dansyl-KF33955 was
covalently bound to the b1, b2, and b5 subunits in the 26S protea-
somes, both in vivo and in vitro. The labeled subunits appear to
have reached maximal levels within 0.5 h, and these levels were
maintained after 1 h of culture, but decreased significantly by 3 h
in the presence of the inhibitor (Fig. 4A and B). When labeling of
subunits in the erythrocyte CP, in which the 19S RP was excluded,
was examined, the kinetics of labeling of all subunits was slower
than that of the 26S proteasome labeling; the maximal labeling re-
quired at least 1 h or more of inhibitor treatment, and the maxi-
mum level of labeled subunits was sustained for 10 h (Fig. 4C).

M. Hasegawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5668–5671
5671
Acknowledgments
Time (h)
0.5
1
3
6 10 16 24
A
B
C
(kDa)
37
We thank Prof. Ryuzo Sasaki for helpful discussions. This re-
search was supported in part by Grants-in-aid from the Japanese
Ministry of Education, Culture, Sports, Science, and Technology
(18790092 to M.H.).
β2
25
20
β1
β5
References and notes
Time (h)
(kDa)
0.5
1
3
6
10 16 24
1. (a) Eytan, E.; Ganoth, D.; Armon, T.; Hershko, A. Proc. Natl. Acad. Sci. U.S.A. 1989,
86, 7751; (b) Baumeister, W.; Walz, J.; Zuhl, F.; Seemuller, E. Cell 1998, 92, 367.
2. Kisselev, A. F.; Goldberg, A. L. Chem. Biol. 2001, 8, 739.
3. (a)Asai,A.;Hasegawa,A.;Ochiai,K.;Yamashita,Y.;Mizukami,T.J.Antibiot.(Tokyo)
2000, 53, 81; (b) Asai, A.; Tsujita, T.; Sharma, S. V.; Yamashita, Y.; Akinaga, S.;
Funakoshi, M.; Kobayashi, H.; Mizukami, T. Biochem. Pharmacol. 2004, 67, 227.
4. Groll, M.; Larionov, O. V.; Huber, R.; de Meijere, A. Proc. Natl. Acad. Sci. U.S.A.
2006, 21, 4576.
β2
25
20
β1
β5
0.5
1
3
6 10 16 24
Time (h)
5. Five hundred nmol of KF33955 and an equivalent amount of the succinimidyl
ester of dansylaminohexanoic acid (Invitrogen Co., Carlsbad, CA) were
(kDa)
37
dissolved in 300 ll of dimethylformamide containing 0.01% triethylamine
β2
and stirred for 16 h at room temperature. Dansyl-KF33955 was purified by
reversed-phase high-performance liquid chromatography. The structure was
supported by ESI–MSMS analysis on an Shimadzu LCMS-IT-TOF. (M++H):
806.370 (calcd 806.372).
25
20
β1
β5
6. Kisselev, A. F.; Goldberg, A. L. Methods Enzymol. 2005, 398, 364.
7. The inhibitors were mixed with 100–200 ng of the human 20S proteasome CP
isolated from human erythrocytes (BIOMOL International L.P.) in enzyme assay
buffer (25 mM HEPES pH 7.6, 0.5 mM EDTA, and 0.03% SDS) in 96-well plates.
SDS was omitted in assays of the trypsin-like activity. The chymotrypsin-like,
trypsin-like, and PGPH catalytic activities were assayed using the fluorogenic
peptide substrates, Suc-LLVY-AMC, Boc-LRR-AMC, and Z-LLE-AMC, respectively
(Peptide Institute Inc., Osaka, Japan). The assays were initiated by addition of
the AMC-substrate. The linear increase in fluorescence was recorded for up to 1
h using a fluorescence plate reader (380 nm excitation, 460 nm emission).
Enzyme activity was estimated using the slope of the fluorescence increase,
and inhibition is represented by IC50 values.
8. HeLa cells were cultured in RPMI 1640 supplemented with 10% heat-
inactivated fetal calf serum and penicillin/streptomycin with 5% CO2. The
viability of HeLa cells was tested using an MTT assay after incubation with
inhibitors added at various concentrations for 48 h. Treatment with (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl) tetrazolium bromide (MTT) was
performed according to the manufacturer’s protocol. Absorbance at 570 nm
was measured using a plate reader.
Figure 4. Western blotting of dansyl-crosslinked subunits of the 26S proteasome in
intact HeLa cells (A) and HeLa cell extract (B) and the purified erythrocyte 20S CP
(C) treated with 1 lM dansyl-KF33955 for the indicated times. The lane indicated
by the bar contains untreated control samples. The proteasome subunit bands
crosslinked to dansyl-KF33955 were detected using an anti-dansyl antibody.
Protein substrates to be degraded enter the CP cavity through a
channel that is opened upon substrate association with the RP
through a mechanism in which Rpt subunits with ATPase activity
mediate the opening.13 Without the 19S RP, the
a ring of the 20S
CP would have no obvious path for substrates to access the active
center chamber of the b ring.14 The in vivo and in vitro labeling of b
subunits in the 26S proteasome, which consists of 20S CP and 19S
RP, is faster than labeling of the isolated CP. This result suggests
that entry of dansyl-KF33955 into the CP cavity may be facilitated
through an RP-induced channel, although the interaction of the
inhibitor with RP components responsible for the channel opening
remains to be verified.
9. Berkers, C. R.; Verdoes, M.; Lichtman, E.; Fiebiger, E.; Kessler, B. M.; Anderson,
K. C.; Ploegh, H. L.; Ovaa, H.; Galardy, P. J. Nat. Methods 2005, 2, 357.
10. HeLa cells were mixed with various concentrations of clasto-lactacystin b-
lactone and MG132 and incubated for 1 h at 37 °C in serum-free medium. Next,
they were mixed with 1.0 lM dansyl-KF33955 and incubated for another hour.
The time-dependent disappearance of labeled subunits in vivo
may result from two events. First, the bound inhibitor may be
gradually released through hydrolysis of the ester bond between
the b lactone ring of the inhibitor and the threonine residue in
the active site. Second, the inhibitor may be unstable under
aqueous conditions so that the free inhibitor concentration can
not be maintained. In concert with this, the inhibitor was grad-
ually degraded, even in purified water, and it was completely
lost overnight, observed in MALDI-TOF MS measurements (data
not shown). The labeled subunits in the 26S proteasome disap-
peared more rapidly (Fig. 4A and B) than those in isolated eryth-
rocyte CP (Fig. 4C). Although further studies are needed to
understand the mechanism underlying this difference, an
intriguing hypothesis is that the RP may stimulate hydrolysis
of the ester bond formed between the inhibitor and threonine
residue, suggesting a novel function of RP in the proteasomal
protein degradation pathway. Alternatively, proteasomes con-
taining subunits whose active sites have been esterified may
be subject to protein degradation.
To stop the assay, the cells were collected by centrifugation (15,000g, 5 min),
treated with denaturing buffer (60 mM Tris–HCl pH 8.8, 5 M urea, 1 M
thiourea, 5 mM EDTA, 1% CHAPS, 1% Triton X-100) at 4 °C, and lysed by
sonication. The samples were separated by SDS–PAGE and protein bands on
the gel were visualized by SYPRO-Red staining (Invitrogen Co.). Images were
acquired using a FLA3000 laser scanner (Fuji Photo Film Co., Ltd., Tokyo, Japan).
Following transfer onto a PVDF membrane, immunodetection using an anti-
dansyl monoclonal antibody (Santa Cruz Biotechnology) was performed. HRP-
conjugated anti-rabbit IgG (MBL) was used as the secondary antibody. Signals
were visualized using an ECL Plus detection kit (Amersham Biosciences) and an
LAS-1000 Plus image analyzer (Fuji Photo Film Co., Ltd.).
11. Extraction of 26S proteasome from HeLa cells was basically performed by the
method reported by Kisselev, A.F; Callard, A; Goldberg, J. Biol. Chem. 2006, 281,
8582. Briefly, HeLa cells were grown up to 50–80% confluency. After harvested
and washed three-times with ice-cold phosphate-buffered saline, the cells
were resuspended in the homogenization buffer (50 mM Tris–HCl pH7.5,
250 mM sucrose, 5 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, and 1 mM
ATP). The cells were permeabilized by the addition of 0.025% digitonin and
incubation on ice for 5 min. The cytosol was ‘‘squeeze out” by centrifugation
for 15 min at 15,000g. The supernatants were used for the next experiments.
12. HeLa cells were mixed with 1.0 lM dansyl-KF33955 and incubated for a given
incubation time at 37 °C in serum-free medium. To stop the assay, the cells
were collected by centrifugation (15,000g, 5 min), treated with the denaturing
buffer. For assays using the extracts 26S proteasome and the purified 20S CP,
solutions of them were mixed with 1.0 lM dansyl-KF33955 for various times.
In this study, we report that a belactosin A derivative labeled
with an immuno-detectable tag, dansyl-KF33955, covalently binds
to the catalytic b-subunits of the proteasome both in vitro and in
vivo. This chemical probe is an excellent tool to study proteasome
function. Moreover, fluorescence detection of the dansyl moiety
may allow in vivo identification of proteasomes with labeled sub-
units and the intracellular fate of the proteasome could thereby be
traced. Optimization of conditions for fluorescence detection is
underway.
The cell extracts and the purified 20S CP solutions were then directly diluted
with 2Â denaturing buffer. The samples were separated by SDS–PAGE.
Following transfer onto a PVDF membrane, immunodetection using the anti-
dansyl monoclonal antibody was performed as described above.10
13. (a) Köhler, A.; Cascio, P.; Leggett, D. S.; Woo, K. M.; Goldberg, A. L.; Finley, D.
Mol. Cell 2001, 7, 1143; (b) Smith, D. M.; Chang, S. C.; Park, S.; Finley, D.; Cheng,
Y.; Goldberg, A. L. Mol. Cell 2007, 27, 731.
14. (a) Groll, M.; Bajorek, M.; Köhler, A.; Moroder, L.; Rubin, D. M.; Huber, R.;
Glickman, M. H.; Finley, D. Nat. Struct. Biol. 2000, 7, 1062; (b) Unno, M.;
Mizushima, T.; Morimoto, Y.; Tomisugi, Y.; Tanaka, K.; Yasuoka, N.; Tsukihara,
T. Structure 2002, 10, 609.