Article

A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism. Nature

Center for Integrated Protein Science at the Department Chemie, Lehrstuhl für Biochemie, Technische Universität München, Lichtenbergstrasse 4, Garching D-85747, Germany.
Nature (Impact Factor: 41.46). 05/2008; 452(7188):755-8. DOI: 10.1038/nature06782
Source: PubMed

ABSTRACT

Pathogenic bacteria often use effector molecules to increase virulence. In most cases, the mode of action of effectors remains unknown. Strains of Pseudomonas syringae pv. syringae (Pss) secrete syringolin A (SylA), a product of a mixed non-ribosomal peptide/polyketide synthetase, in planta. Here we identify SylA as a virulence factor because a SylA-negative mutant in Pss strain B728a obtained by gene disruption was markedly less virulent on its host, Phaseolus vulgaris (bean). We show that SylA irreversibly inhibits all three catalytic activities of eukaryotic proteasomes, thus adding proteasome inhibition to the repertoire of modes of action of virulence factors. The crystal structure of the yeast proteasome in complex with SylA revealed a novel mechanism of covalent binding to the catalytic subunits. Thus, SylA defines a new class of proteasome inhibitors that includes glidobactin A (GlbA), a structurally related compound from an unknown species of the order Burkholderiales, for which we demonstrate a similar proteasome inhibition mechanism. As proteasome inhibitors are a promising class of anti-tumour agents, the discovery of a novel family of inhibitory natural products, which we refer to as syrbactins, may also have implications for the development of anti-cancer drugs. Homologues of SylA and GlbA synthetase genes are found in some other pathogenic bacteria, including the human pathogen Burkholderia pseudomallei, the causative agent of melioidosis. It is thus possible that these bacteria are capable of producing proteasome inhibitors of the syrbactin class.

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LETTERS
A plant pathogen virulence factor inhibits the
eukaryotic proteasome by a novel mechanism
Michael Groll
1
*, Barbara Schellenberg
2
*, Andre
´
S. Bachmann
3,4
, Crystal R. Archer
3,4
, Robert Huber
5,6,7
,
Tracy K. Powell
8
, Steven Lindow
8
, Markus Kaiser
9
& Robert Dudler
2
Pathogenic bacteria often use effector molecules to increase viru-
lence. In most cases, the mode of action of effecto rs remains
unknown. Strains of Pseudomonas syringae pv. syringae (Pss)
secrete syringolin A (SylA), a product of a mixed non-ribosomal
peptide/polyketide synthetase, in planta
1
. Here we identify SylA as
a virulence factor because a SylA-negative mutant in Pss strain
B728a obtained by gene disruption was markedly less virulent
on its host, Phaseolus vulgaris (bean). We show that SylA irrevers-
ibly inhibits all three catalytic activities of eukaryotic protea-
somes, thus adding proteasome inhibition to the repertoire of
modes of action of virulence factors. The crystal structure of the
yeast proteasome in complex with SylA revealed a novel mech-
anism of covalent binding to the catalytic subunits. Thus, SylA
defines a new class of proteasome inhibitors that includes glido-
bactin A (GlbA), a structurally related compound from an
unknown species of the order Burkholderiales
2
, for which we
demonstrate a similar proteasome inhibition mechanism. As pro-
teasome inhibitors are a promising class of anti-tumour agents,
the discovery of a novel family of inhibitory natural products,
which we refer to as syrbactins, may also have implications for
the development of anti-cancer drugs
3
. Homologues of SylA and
GlbA synthetase genes are found in some other pathogenic bac-
teria, including the human pathogen Burkholderia pseudomallei,
the causative agent of melioidosis
4
. It is thus possible that these
bacteria are capable of producing proteasome inhibitors of the
syrbactin class.
Pss is a pathogen of many plant species, causing, for example,
brown spot disease on bean (P. vulgaris )
5
. Some Pss strains secrete
SylA, a peptide derivative synthesized by a mixed non-ribosomal
peptide synthetase (NRPS)/polyketide synthetase (PKS) encoded
by a gene cluster which was previously cloned from the strain Pss
B301D-R
2
. To examine the functional role of SylA, we constructed a
SylA-negative mutant by targeted gene disruption of the sylC gene in
Pss B728a, a strain that causes brown spot disease on bean (P. vul-
garis) and whose SylA synthetase gene cluster is almost identical to
that of strain B301D-R
2
.ThesylC gene encodes the first NRPS
module of the SylA synthetase, and disruption of sylC in the B301D-
R strain was shown to abolish SylA synthesis
2
. Infectionexperimentson
bean plants revealed a significantly reduced virulence of the mutant
strain. The number of brown spots developing on bean leaves after
spray inoculation was reduced to 29 6 21% compared with the wild
type in four independent experiments (Fig. 1). Thus, SylA was iden-
tified as a virulence factor in this plant–pathogen interaction.
Previously performed transcript profiling revealed that exogenous
application of SylA on wheat and Arabidopsis thaliana leads to pro-
nounced changes in global gene activity
6
. Intriguingly, transcripts
encoding all proteasome subunits and many heat-shock proteins
accumulated in Arabidopsis leaves. Similar observations were reported
for yeast and mammalian cells after treatment with proteasome
inhibitors
7,8
. Therefore, we tested whether SylA was a proteasome
inhibitor. The eukaryotic 20S proteasome contains three catalytic
subunits (b1, b2andb5) conferring caspase-like, trypsin-like and
chymotrypsin-like proteolytic activities, respectively
3
. In vitro experi-
ments showed that SylA inhibited all of them (Fig. 2a–d). In addition,
measurement of proteasome activity in the presence of SylA revealed
that the proteolytic reaction velocity was not constant but diminished
as a function of time. This is similar to what is observed in the presence
of the irreversible proteasome inhibitor epoxomicin and in contrast
to the effect of the reversible peptide aldehyde inhibitor MG-132
(Fig. 2a), suggesting that SylA acts as an irreversible proteasome inhi-
bitor. This finding was confirmed by removing inhibitors and sub-
strate from chymotrypsin-like cleavage reactions by dialysis. Addition
of substrate to dialysed proteasomes previously inhibited by MG-132
led to renewed activity, whereas proteasomes that had been inhibited
by epoxomicin or SylA remained inactive (Supplementary Fig. 1). The
chymotrypsin-like activity was found to be most sensitive to SylA
inhibition, whereas higher concentrations of SylA were necessary to
*These authors contributed equally to this work.
1
Center for Integrated Protein Science at the Department Chemie, Lehrstuhl fu¨r Biochemie, Technische Universita
¨t
Mu¨nchen, Lichtenbergstrasse 4, Garching D-85747, Germany.
2
Zurich
Basel Plant Science Center, Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland.
3
Cancer Research Center of Hawaii, University of
Hawaii at Manoa, 1236 Lauhala Street, Honolulu, Hawaii 96813, USA.
4
Cell and Molecular Biology Graduate Program, John A. Burns School of Medicine, University of Hawaii at Manoa,
651 Ilalo Street, Honolulu, Hawaii 96813, USA.
5
Max-Planck-Institut fu¨r Biochemie, D-82152 Martinsried, Germany.
6
School of Biosciences, Cardiff University, Cardiff CF10 3US, UK.
7
Zentrum fu¨r medizinische Biotechnologie, Universita
¨
t Duisburg-Essen, D-45117 Essen, Germany.
8
Department of Plant and Microbial Biology, University of California, 111 Koshland
Hall, Berkeley, California 94720-3102, USA.
9
Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany.
0
40
80
120
160
OMOMOO
Lesions per trifoliate leaf
Exp. 1 Exp. 2 Exp. 3
Exp. 4
*
**
**
**
**
**P < 0.01
*P < 0.05
Wild type
sylC KO
Figure 1
|
Syringolin-negative mutant exhibits reduced virulence. Five pots
per experiment (Exp), each with eight 18-day-old bean plants, were spray-
inoculated with 10
5
cells per millilitre of wild-type or SylA-negative (sylC
KO) strains of Pss B728a. Lesion numbers per trifoliate leaf were counted on
the oldest (O) and middle-aged (M) leaves. Mean lesion numbers 6 s.d. over
the five replica pots are given. p, error probability (two-sided t-test).
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inhibit trypsin-like and caspase-like activities (Fig. 2b–d). In contrast
to MG-132, which also inhibits cysteine proteases, SylA inhibition of
the proteasome was specific because both papain (Fig. 2e) and the
serine protease trypsin were not affected by SylA, even at significantly
higher concentrations (up to 500 mM). SylA also inhibited the
chymotrypsin-like activity of the proteasome in crude extracts of
etiolated bean seedlings (Supplementary Fig. 2).
We next determined whether SylA inhibits in vivo proteasome
function in Arabidopsis seedlings and human cultured cells. To this
end, an Arabidopsis line homozygous for a CyclinB1;1::uidA (GUS)
reporter fusion gene
9
was used. The mitotic CyclinB1;1 gene is
expressed only around the G2/M transition of the cell cycle
10
. The
fusion gene contains a cyclin destruction box leading to the pro-
teasomal degradation of the protein at the end of the M phase
11
.
Proteasomal dysfunction due to a mutation has been shown to result
in strong GUS staining of dividing cells
12
. Indeed, much stronger
staining of dividing cells in the root meristem of 3-day-old seedlings
treated with 10 mM SylA solution compared with controls was
observed (Supplementary Fig. 3). Incubation of human SK-N-SH
neuroblastoma cells with SylA for 2 h resulted in a dose-dependent
decrease of proteasome activity (Supplementary Fig. 4a). As
expected
13
, incubation of SK-N-SH cells with 25 mM SylA resulted
in a time-dependent accumulation of ubiquitinated proteins (Sup-
plementary Fig. 4d). The SylA treatment of SK-N-SH cells was
further accompanied by a time-dependent increase in the protein
levels of the tumour suppressor protein p53, a known target of the
proteasome and an important regulator of apoptosis and cell-cycle
progression (Supplementary Fig. 4d).
To elucidate the binding mode of SylA to the proteasome we co-
crystallized SylA with the yeast 20S proteasome and collected data to
2.9 A
˚
resolution. Crystallographic refinement started from the coor-
dinates of the yeast 20S proteasome
14
followed by anisotropic overall
temperature factor correction and positional refinement using the
Crystallography & NMR system (CNS) software suite (R
crys
/R
free
5
21.8/24.8; Supplementary Table 1)
15
. Electron-density maps calcu-
lated with phases after twofold averaging allowed a detailed inter-
pretation of SylA (Fig. 3c), revealing that SylA covalently binds to
the hydroxy group of the active site amino (N)-terminal threonine
by a novel mechanism: Thr1O
c
of the proteasome performs a
Michael-type 1,4-addition to the double bond located at C4 in the
12-membered ring system of the inhibitor. The resulting covalent
ether bond with the 12-membered ring system causes the irreversible
inhibition. This reaction is facilitated by Gly47N, which stabilizes the
carbonyl anion of SylA in its activated transition intermediate state
by hydrogen bonds (oxyanion hole) (Fig. 3b). Furthermore, the 12-
membered ring structure of SylA containing the functional reactive
group has a constrained conformation favouring high-affinity bind-
ing for entropic reasons compared with flexible ligands (Fig. 3e). The
functional reactive double bond at the 12-membered ring is com-
bined with a dipeptide located in its close proximity (Fig. 3a). This
dipeptide is essential for stabilization of SylA by formation of an
antiparallel b-sheet at the substrate-binding channel, which increases
the mean residence time of the inhibitors at the active centre for
completing the covalent ether bond formation with Thr1O
c
.
The peptide structure of SylA is closely related to glidobactin A
(GlbA; Fig. 3a), an acylated peptide derivative reported to have anti-
fungal and anti-tumour activity
16
. Thus, we tested GlbA isolated from
strain K481-B101 (ATCC 53080; DSM 7029)
4
for proteasome inhibi-
tion. Indeed, GlbA blocked the chymotrypsin-like activity irrever-
sibly at low concentrations, whereas the trypsin-like activity was less
sensitive (Fig. 2d) and the caspase-like activity was not inhibited at
the concentrations tested (up to 20 mM). The crystal structure of
GlbA in complex with the proteasome was elucidated at 2.7 A
˚
resolution, yielding R
crys
/R
free
5 23.4/26.2 (Supplementary Table 1)
(Fig. 3d). As expected, the complex structure revealed the same
mechanism of inactivation of the chymotrypsin-like (Fig. 3f) and
trypsin-like activities as SylA, whereas GlbA did not bind to the
caspase-like active site.
The results described above show that SylA is a virulence factor
that inhibits proteasome function, thus adding proteasome inhibi-
tion to the repertoire of modes of action of such factors. Although
many virulence-related factors are delivered into host cells by type III
secretion systems
17–19
, SylA is thought to be secreted by an exporter
belonging to the major facilitator superfamily
2
. This, together with
the fact that exogenous application of SylA leads to proteasome
inhibition (this report), inhibition of cell proliferation and induction
of apoptosis
20
in mammalian cancer cells, as well as changes in gene
activity typical for proteasome inhibition in Arabidopsis and wheat,
0 20406010 30 50
Time (min)
8.0 × 10
4
1.0 × 10
5
1.2 × 10
5
1.4 × 10
5
1.6 × 10
5
1.8 × 10
5
RFU
Control
25 µM MG132
25 µM SylA
50 µM MG132
100 µM MG132
50 µM SylA
100 µM SylA
Inhibitor Inhibited activity K
i
k
association
Chymotrypsin-like
843 ± 8.4 nM (n = 3) 863 ± 106 M
–1
s
–1
(n = 6; 100–200 nM)
Trypsin-like
6.7 ± 0.7 µM (n = 6) 94 ± 12 M
–1
s
–1
(n = 6; 150–600 nM)
SylA
Caspase-like n.d.
6 ± 0.3 M
–1
s
–1
(n = 6; 20–40 µM)
Chymotrypsin-like
49 ± 5.4 nM (n = 3) 3,377 ± 341 M
–1
s
–1
(n = 6; 40–60 nM)
GlbA
Trypsin-like
2.0 ± 0.6 µM (n = 6) 141 ± 21 M
–1
s
–1
(n = 6; 250–500 nM)
Chymotrypsin-like activity
16,000
12,000
8,000
4,000
0
0 20 40 60 80 100 120
Time (min)
Control
1.6 µM MG132
1.6 µM Epo
0.2 µM Epo
0.2 µM SylA
1.6 µM SylA
RFU
a
Trypsin-like activity
0 20 40 60 80 100 120
1,600
1,400
1,200
1,000
800
600
Time (min)
Control
1.25 µM SylA
5 µM SylA
40 µM SylA
RFU
b Caspase-like activity
0 20406080
100
120
Time (min)
1,000
2,000
3,000
4,000
Control
5 µM SylA
20 µM SylA
80 µM SylA
RFU
c
e
d
Papain
Figure 2
|
SylA inhibits the eukaryotic proteasome. ac, Inhibition of
chymotrypsin-like
(a), trypsin-like (b) and caspase-like (c) activities of
mammalian proteasomes by SylA, epoxomicin (Epo) and MG-132 using
fluorogenic substrates. RFU, relative fluorescence units.
d, Apparent K
i
9
values and rates of covalent inhibition (k
association
) over inhibitor
concentrations given in parentheses were derived from plots as shown in
ac (see Methods). Values represent means 6 s.d. e, Cleavage of fluorescent
casein by papain in the presence of MG-132 and SylA.
LETTERS NATURE
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indicates that SylA is taken up by cells. As SylA is a hydrophilic
molecule, it probably cannot passively cross membranes, and
thus an uptake transporter must be postulated, which remains to
be identified.
Previous studies have shown that the host proteasome has been
instrumentalized by plant pathogens to suppress host defence reac-
tions by type III effectors mediating proteasomal degradation of
specific host proteins
2,21,22
. On the other hand, there is increasing
evidence that the ubiquitin–proteasome degradation pathway is
essential for pathogen defence and disease immunity of plants
23,24
.
Pss appears to capitalize on this by inhibiting the host proteasome
using SylA, thereby suppressing host defence reactions directly or
indirectly. It also remains to be seen whether the proteasome is the
sole target of SylA.
A unique model completely explaining the synthesis of the
peptide part of SylA, including the functionally important double
bond at C4 of the 12-membered ring system, has been proposed
based on the architecture of the sylC and sylD genes in the SylA
synthetase gene cluster
2
. The model allowed the recent cloning of
genes responsible for GlbA synthesis, including the sylC and sylD
homologues glbF and glbC, from K481-B101, the strain from which
GlbA was originally isolated
16
and which belongs to an unknown
species of the order Burkholderiales
4
. A National Center for
Biotechnology Information database search of all sequenced bacterial
genomes revealed homologues of these genes in the insect pathogen
Photorhabdus luminescens and the human pathogen B. pseudomallei ,
the causative agent of melioidosis
4
. These organisms are therefore
hypothesized to be capable of synthesizing proteasome inhibitors
of the syrbactin class. If this prediction is confirmed, it will be inter-
esting to determine whether the respective compounds are involved
in virulence.
Proteasome inhibitors form a promising new therapeutic class of
drugs against cancer and other diseases, causing intense interest in
such molecules
3,25
. Indeed, SylA was recently shown to inhibit pro-
liferation and induce apoptosis in neuroblastoma and ovarian cancer
cells
20
. Elucidation of the structural inhibition motif of syrbactins
opens new perspectives in the identification of natural products with
proteasome inhibition potential, as well as the design and creation of
new specific, selective and efficient proteasomal inhibitors. New inhi-
bitors derived from such natural products can find their potential
application in drug development.
METHODS SUMMARY
Mutant construction and plant inoculations. Construction of the SylA-
negative mutant by plasmid insertion into the sylC gene of Pss B728a was as
described for the corresponding mutant in strain Pss B301D-R
2
. Virulence of Pss
c
d
e
f
Syringolin A
a
b
Glidobactin A
Figure 3
|
Structural basis for proteasome inhibition by syrbactins.
a
, Chemical structure of SylA and GlbA. Red, a,b-unsaturated carbonyl
group reacting with Thr1O
c
; green, dipeptide bond stabilizing the inhibitor
upon proteasome binding; blue, molecule part determining active site
specificity; yellow, aliphatic tail of GlbA.
b, Mechanism of binding of SylA/
GlbA to the active site Thr1.
c, d, Stereo representation of the chymotryptic-
like active site (rose, subunit b5; light blue, subunit b6) in complex with
(
c) SylA (green; PDB accession code 2ZCY) and (d) GlbA (green, aliphatic
tail in yellow; PDB accession code 3BDM). Magenta, covalent linkage of
inhibitors with active site Thr1; dotted lines indicate hydrogen bonds. Black,
residues performing specific interactions with SylA and GlbA. Electron-
density maps (grey) are contoured from 1s in similar orientations around
Thr1.
e, Electrostatic potential surface (contoured from 115kT/e (intense
blue) to 215kT/e (intense red)) of SylA bound to subunit b5.
f, Structural
superposition of SylA (green) with GlbA (yellow) bound to subunit b5.
NATURE
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strains was assessed by counting the number of lesions on leaves of bean (P.
vulgaris) plants sprayed with suspensions of 10
5
cells per millilitre (ref. 26).
Isolation of SylA and GlbA. SylA and GlbA have been isolated as described
2,4,27
.
SylA solutions with defined concentrations were prepared by dissolving
weighted amounts of SylA in water. Concentrations of GlbA solutions were
determined by measuring the absorption at 261 nm in methanol using an extinc-
tion coefficient of 35,000 M
21
cm
21
(ref. 16). For proteasome inhibition assays,
stock solutions of GlbA in DMSO were diluted with water.
Proteasome/protease inhibition assays. In vitro proteasome inhibition assays
were performed in 96-well microtiter plates with human erythrocyte 20S pro-
teasomes using the Assay Kit for Drug Discovery AK-740 (Biomol). In vivo
proteasome inhibition in cultured mammalian cells was measured using the
proteasome-Glo cell-based luminescent assay (Promega) in solid white 96-well
microtiter cell culture plates. Bovine pancreas trypsin (Pierce) and Papaya latex
papain (Sigma-Aldrich) inhibition assays were performed with the SensoLyte
Green Protease Fluorometric Assay Kit (AnaSpec).
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 10 October 2007; accepted 28 January 2008.
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Supplementary Informat ion is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank U. Grossniklaus and J. Celenza (Boston University)
for the CycB1;1-GUS line. U. Grossniklaus and H. Gehring are thanked for reading the
manuscript. We also thank D. Albert, H. Gehring, Z. Hasenkamp, R. Go, D. Koomoa,
J. Molnar, A. Niewienda and C. Wallick for technical advice and assistance. We are
grateful to L. Eberl for use of equipment. C.R.A was supported by a graduate
student research assistantship from the Cell and Molecular Biology Graduate
Program, University of Hawaii. Support by grants from the Swiss National Science
Foundation to R.D. is acknowledged.
Author Contributions M.K., M.G., S.L., R.D. and A.S.B. designed experiments,
analysed results and wrote the manuscript. R.D. constructed the sylA-negative
mutant, T.K.P. performed the bean inoculation experiments, B.S. isolated SylA and
GlbA, and performed in vitro proteasome and protease inhibition assays. M.G., M.K
and R.H. performed and analysed crystallization experiments. C.R.A. performed the
mammalian cell-based proteasome inhibition assays and immunoblot studies.
Author Information Atomic coordinates have been deposited in the RCSB Protein
Data Bank (http://www.rcsb.org/pdb) under the accession code 2ZCY (yeast 20S
proteasome
syringolin A complex) and 3BDM (yeast 20S
proteasome
glidobactin A complex). Reprints and permissions information is
available at www.nature.com/reprints. Correspondence and requests for
materials should be addressed to R.D. (rdudler@botinst.uzh.ch), A.S.B.
(abachmann@crch.hawaii.edu) or M.K. (markus.kaiser@cgc.mpg.de).
LETTERS NATURE
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Vol 452
|
10 April 2008
758
Nature
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Page 4
METHODS
In vitro proteasome/protease assays and determination of constants.
Proteasome inhibition assays with human erythrocyte 20S proteasomes (AK-
740, Biomol) were performed at 37 uC in 100 ml reaction volumes containing
2 mgml
21
20S proteasome and 100 mM Suc-LLVY-AMC, Boc-LRR-AMC or
Z-LLE-AMC for assaying the chymotrypsin-like, trypsin-like and caspase-like
activity, respectively. Fluorescence was monitored with an MWGt Sirius HT
plate reader (BIO-TEK Instruments) equipped with 360 nm excitation and
460 nm emission filters.
Fluorescence values (RFU) measured in proteasome inhibition assays were
plotted versus time (t). The fluorescence data (see Fig. 2) were fitted by the least
squares method using SigmaPlot version 10.0 software (Systat Software) to the
equation f 5 f
0
1 v
s
t 1 [(v
i
2 v
s
)/k
obs
][1 2 exp(2k
obs
t)], where v
i
and v
s
are
initial and final velocities, respectively, and k
obs
is the pseudo-first-order asso-
ciation rate constant
13,28
. Fittings with R
2
values . 0.99 were usually obtained.
The rate of covalent inhibition (k
association
) which equals k
obs
/[I], was calculated
over the range of inhibitor concentrations given in parentheses in Fig. 2d. The
initial velocities (v
i
) were determined from the time derivative of these curves
and plotted against the corresponding inhibitor concentration ([I]). Where
possible, apparent K
i
9 values were determined by plotting v
i
against the inhibitor
concentration ([I]). Using SigmaPlot, the data were fitted to the hyperbolic
equation v
i
5 c 1 [v
0
/(K
i
91[I])], where v
0
is the velocity of the control.
Usually, R
2
values . 0.98 were obtained.
Bovine pancreas trypsin (Pierce) and Papaya latex papain (Sigma-Aldrich)
inhibition assays were performed with the SensoLyteTM Green Protease
Fluorometric Assay Kit (AnaSpec) in 100 ml volumes containing 200 ng ml
21
enzyme at 33 uC. For detection, 485/550 nm excitation/emission filters were
used.
Inhibition assays of plant proteasomes were performed with crude lysates of
7-day-old etiolated bean seedlings. Hypocotyls and emerging first leaves of
seedlings were ground in liquid nitrogen and extracted with twice the amount
(v/w) of extraction buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 100 mM
NaCl, 10 mM sodium orthovanadates). After centrifugation at 500g for
15 min, the supernatant was used for inhibition experiments of the proteasome
chymotrypsin-like activity in microtitre plates. Each reaction contained 20 ml
lysate (about 100 mg protein), 100 mM Suc-LLVY-AMC substrate, desired con-
centrations of inhibitors and assay buffer (AK-740, Biomol) to a volume of
100 ml. In some experiments, the cysteine protease inhibitor E64 (Sigma) was
included in each reaction at a final concentration of 50 mM. Reactions were
monitored at room temperature every 4 min as described above. Reaction rates
were approximately linear between the 8 and 32 min time points.
In vivo proteasome inhibition and immunoblot analysis. Source and main-
tenance of the human neuroblastoma cell line SK-N-SH have been described
20,29
.
For in vivo proteasome inhibition assays, wells of solid white 96-well microtiter
cell culture plates were seeded with 1.1 3 10
5
cells per millilitre (90 ml per well).
After 24 h, 10 ml SylA was added at different concentrations (0–100 mM) and
incubated for 2 h. The proteasome inhibitor epoxomicin (0–1 mM) and the
apoptosis-inducing agent cerulenin (0–50 mgml
21
)
30,31
were used as positive
and negative controls, respectively (Supplementary Fig. 4). Microtiter plates
were then equilibrated to room temperature for 15 min before 100 mlof
proteasome Glo reagent (containing the bioluminescent substrate Suc-LLVY-
aminoluciferin) per well was added and luminescence measured according to the
manufacturer’s instructions (Promega).
For immunoblot analysis, cells were seeded in six-well cell culture plates at a
concentration of 3.8 3 10
5
cells per millilitre (1.98 ml per well). After 24 h, 20 ml
of SylA (final concentration 25 mM) or cell culture medium (control, C) was
added. For time-course experiments, cell treatments were staggered to process all
samples at the same time after 24 h. Cell lysates were prepared as previously
reported
29
. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE), electro-transfer to PVDF Immobilon-P membranes (Millipore)
and antibody incubations were performed according to standard procedures.
The primary antibodies used were rabbit whole serum ubiquitin (1:150) (U5379;
Sigma), mouse monoclonal tumour suppressor protein p53 (1:250) (sc-126;
Santa Cruz Biotechnology) and rabbit monoclonal a-tubulin (1:1,500) (11H10;
Cell Signalling Technology). Horseradish peroxidase (HRP)-conjugated anti-
mouse or anti-rabbit antibodies (1:5,000) were used as secondary antibodies.
Membranes were developed using the ECL Plus kit following the manufacturer’s
protocol (Amersham Biosciences) and exposed to Blue Lite Autorad Film (ISC
BioExpress). Membranes were stripped with ECL stripping buffer (62.5 mM Tris-
HCl, pH 6.7, 2% SDS, 100 mM b-mercaptoethanol) for 20 min at 55 uC and
sequentially re-probed with the next antibody. The experiment was repeated three
times with similar results, as shown in Supplementary Fig. 4d.
Treatment and GUS-staining of transgenic Arabidopsis carrying a CyclinB1;
1::uidA fusion gene. A homozygous line containing a transgene derived from
pCDG in which the CycB1;1 promoter plus the sequence encoding the first 150
amino acids of the CYCB1;1 protein including a cyclin destruction box were
fused in frame to the uidA reporter gene encoding b-glucuronidase (GUS)
9
.
Seeds of this line, which were provided by U. Grossniklaus, University of
Zurich, were germinated on vertically oriented MS plates (4.4 g l
21
Murashige
and Skoog basal medium with Gamborg’s vitamins (Sigma), 3% sucrose and
0.6% Phytagel (Sigma)) that were incubated under a 16 h light, 8 h dark regime at
22 uC. Five-microlitre droplets of water with or without 10 mM SylA were applied
to 3-day-old seedlings. After an 18 h incubation period, seedlings were trans-
ferred to a 96-well microtiter plate, stained for GUS activity for 60 min as
described
32
and observed under a Leica DMR microscope.
Co-crystallization. Crystals of the 20S proteasome from S. cerevisiae were
grown in hanging drops at 24 uC as described
14,33
and incubated for 60 min with
SylA or GlbA. The protein concentration used for crystallization was 40 mg ml
21
in Tris-HCl (10 mM, pH 7.5) and EDTA (1 mM). The drops contained 3 mlof
protein and 2 ml of the reservoir solution (30 mM magnesium acetate, 100 mM
morpholino-ethane-sulphonic acid (pH 7.2) and 10% MPD).
The space group belongs to P2
1
with cell dimensions of about a 5 134 A
˚
,
b 5 302 A
˚
, c 5 144 A
˚
and b 5 112u (see Supplementary Table 1). Data to 2.9 A
˚
for the yeast 20S proteasome–SylA- and to 2.7 A
˚
for the 20S proteasome–GlbA
complexes were collected using synchrotron radiation with l 5 1.05 A
˚
on
the BW6-beamline at DESY, Hamburg, Germany, and with l 5 1.0 A
˚
on the
X06SA-beamline at SLS, Villingen, Switzerland, respectively. Crystals were
soaked in a cryoprotecting buffer (30% MPD, 20 mM magnesium acetate,
100 mM morpholino-ethane-sulphonic acid, pH 6.9) and frozen in a stream
of liquid nitrogen gas at 90 K (Oxford Cryo Systems). X-ray intensities were
evaluated by using the DENZO program package
34
and data reduction was
performed with CCP4
35
. The anisotropy of diffraction was corrected by an
overall anisotropic temperature factor by comparing observed and calculated
structure amplitudes using the program CNS
15
. Electron density was improved
by averaging and back transforming the reflections ten times over the twofold
non-crystallographic symmetry axis using the program package MAIN
36
.
Conventional crystallographic rigid body, positional and temperature factor
refinements were performed with CNS
15
using the yeast 20S proteasome struc-
ture as starting model
14
. Model building was performed using the program
MAIN. Apart from the bound inhibitor molecules, structural changes were only
noted in the specificity pockets. Temperature factor refinement indicates full
occupancies of all inhibitor-binding sites. The inhibitors have been omitted for
phasing.
28. Fenteany, G. et al. Inhibition of proteasome activities and subunit-specific amino-
terminal threonine modification by lactacystin. Science 268, 726
731 (1995).
29. Wallick, C. J. et al. Key role for p27(Kip1), retinoblastoma protein Rb, and MYCN in
polyamine inhibitor-induced G(1) cell cycle arrest in MYCN-amplified human
neuroblastoma cells. Oncogene 24, 5606
5618 (2005).
30. Geerts, D. et al. Expression of PRA1 domain family, member 2 (PRAF2) in
neuroblastoma: correlation with clinical features, cellular localization, and
cerulenin-mediated apoptosis regulation. Clin. Cancer Res. 13, 6312
6319 (2007).
31. Heiligtag, S. J., Bredehorst, R. & David, K. A. Key role of mitochondria in cerulenin-
mediated apoptosis. Cell Death Differ. 9, 1017
1025 (2002).
32. Rodrigues-Pousada, R. A. et al. The Arabidopsis 1-aminocyclopropane-1-
carboxylate synthase gene-1 is expressed during early development. Plant Cell 5,
897
911 (1993).
33. Groll, M. & Huber, R. Purification, crystallization and X-ray analysis of the yeast
20S proteasomes. Methods Enzymol. 398, 329
336 (2005).
34. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in
oscillation mode. Methods Enzymol. 276, 307
326 (1997).
35. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface to
the CCP4 program suite. Acta Crystallogr. D 59, 1131
1137 (2003).
36. Turk, D. Improvement of a Programme for Molecular Graphics and Manipulation of
Electron Densities and Its Application for Protein Structure Determination. PhD thesis,
Technische Univ. Mu¨nchen (1992).
doi:10.1038/nature06782
Nature
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Page 5
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