proteome-wide identification of mycobacterial pupylation targets
TRANSCRIPT
REPORT
Proteome-wide identification of mycobacterialpupylation targets
Christian Poulsen1,4, Yusuf Akhter1,4, Amy Hye-Won Jeon2, Gerold Schmitt-Ulms2, Helmut E Meyer3, Anja Stefanski3, Kai Stuhler3,Matthias Wilmanns1,* and Young-Hwa Song1,*
1 EMBL-Hamburg Unit, European Molecular Biology Laboratory, Hamburg, Germany, 2 Centre for Research in Neurodegenerative Diseases, University of Toronto,Toronto, Ontario, Canada and 3 Medizinisches Proteom-Center, Ruhr Universitat Bochum, Universitatsstra�e, Bochum, Germany4 These authors contributed equally to this work* Corresponding authors. EMBL-Hamburg Unit, European Molecular Biology Laboratory, Notkestrasse 85, 22603 Hamburg, Germany. Tel.: þ 49 40 89902 126;Fax: þ 49 40 89902 149; E-mail: [email protected], [email protected]
Received 13.11.09; accepted 12.5.10
Mycobacteria use a unique system for covalently modifying proteins based on the conjugation of asmall protein, referred to as prokaryotic ubiquitin-like protein (PUP). In this study, we report aproteome-wide analysis of endogenous pupylation targets in the model organism Mycobacteriumsmegmatis. On affinity capture, a total of 243 candidate pupylation targets were identified by twocomplementary proteomics approaches. For 41 of these protein targets, direct evidence for a total of48 lysine-mediated pupylation acceptor sites was obtained by collision-induced dissociation spectra.For the majority of these pupylation targets (38 of 41), orthologous genes are found in theM. tuberculosis genome. Interestingly, approximately half of these proteins are involved inintermediary metabolism and respiration pathways. A considerable fraction of the remainingtargets are involved in lipid metabolism, information pathways, and virulence, detoxification andadaptation. Approximately one-third of the genes encoding these targets are located in seven geneclusters, indicating functional linkages of mycobacterial pupylation targets. A comparison of thepupylome under different cell culture conditions indicates that substrate targeting for pupylation israther dynamic.Molecular Systems Biology 6:386; published online 13 July 2010; doi:10.1038/msb.2010.39Subject Categories: proteomics; microbiology & pathogensKeywords: covalent modification; gene clustering; mycobacteria; proteomics; pupylation
This is an open-access article distributed under the terms of the Creative Commons AttributionNoncommercial No Derivative Works 3.0 Unported License, which permits distribution and reproductioninanymedium,providedtheoriginalauthorandsourcearecredited.This licensedoesnotpermitcommercialexploitation or the creation of derivative works without specific permission.
Introduction
The posttranslational modification of proteins by ubiquitin-likemodifiers has attracted much interest in recent years. Ubiquitinconjugation, originally linked to protein degradation by theproteasome, is known to control many kinds of biologicalfunction and physiological activity (Hershko and Ciechanover,1986, 1998; Crosas et al, 2006; Grabbe and Dikic, 2009).Recently, a small protein (Rv2111c) from Mycobacteriumtuberculosis has been described as a prokaryotic ubiquitin-likeprotein (PUP) (Pearce et al, 2008; Burns et al, 2009), constitutingthe first known ubiquitin-like system in bacteria. The PUP-related genes are confined to the actinobacteria phylum and arefrequently observed in proximity to genes encoding proteincomponents of the proteasome (Iyer et al, 2008).
To date, two proteins have been identified as part of thePUP conjugation machinery, DOP (Rv2112c) and PafA (Rv2097c),possessing deamidase and putative PUP ligase activities, respec-tively (Striebel et al, 2009). In addition, three potential mycobac-terial PUP substrates have been found (Pearce et al, 2008; Burnset al, 2009): (1) malonyl coenzyme A acyl carrier protein (FabD)that is involved in cell wall biosynthesis (Kremer et al, 2002); (2)myoinositol-1-phosphate synthase (MIPS) that catalyzes the firstreaction step of the biosynthesis pathway of L-myo-inositol-1-phosphate (Stieglitz et al, 2005); and (3) superoxide dismutase(SOD) that has a key part in the cellular detoxification ofsuperoxide anions (Kusunose et al, 1976). However, it is notknown whether the restricted number of reported targets reflects acurrent limitation in their detection, or is the result of their relativetransience, due to their rapid proteasomal degradation.
Molecular Systems Biology 6; Article number 386; doi:10.1038/msb.2010.39Citation: Molecular Systems Biology 6:386& 2010 EMBO and Macmillan Publishers Limited All rights reserved 1744-4292/10www.molecularsystemsbiology.com
& 2010 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2010 1
To explore the impact of pupylation as a new type offunctional regulation in mycobacterial biology, it is essential toexplore to what extent components of the mycobacterialproteome are targeted for pupylation. In this study, we usedthe endogenous PUP conjugation/processing machinery fromM. smegmatis to identify potential PUP targets from enrichedpurified material. Using two complementary proteomicsapproaches, we have observed a total of 48 pupylation sitesin 41 mycobacterial substrates. The majority of these targetsare encoded by gene loci clustered in restricted regions of theM. smegmatis genome.
Results and discussion
Identification of PUP substrates from theM. smegmatis proteome
To provide insight into the overall properties of PUP, we firstexpressed the M. tuberculosis protein (Rv2111c, mtPUP) inEscherichia coli and purified it to homogeneity (Figure 1A).Further biophysical characterization is described in theSupplementary information.
To identify potential PUP substrates, we also expressedN-terminally poly-histidine-tagged mtPUP in M. smegmatis,which is a well-established model system (Hatfull et al, 2008).Affinity-purified mtPUP gave rise to multiple bands whenassessed by SDS–PAGE (Figure 1B, Supplementary Figure 1Aand B) and by western blot analysis (Supplementary Figure1C). When using a mtPUP(Q64A) variant the multiple bandpattern was lacking, indicating that it is caused by specificpupylation involving Gln64 of PUP. The same result wasobtained when using a M. tuberculosis control target (Rv3874).Supporting this observation, mtPUP heterologously expressedin E. coli also migrated as a single band after nickel-nitrilotriacetic acid (NiNTA) affinity purification, showingthat the effect is specific to M. smegmatis (Figure 1A).
We first used in-solution digestion followed by two-dimensional (2D) liquid chromatography, both by offlinestrong cation exchange (SCX) followed by online reversed
phase (RP), and by electrospray ionization (ESI) tandem massspectrometry (MS/MS), using a quadrupole-time-of-flightinstrument (QqTOF). The aim of this approach was to obtaina high coverage of potential pupylation targets. NiNTA eluateprotein fractions from M. smegmatis served as source materialfor this analysis. To minimize co-enrichment of indirectbinders, the purification was carried out under denaturingconditions. To permit the recognition of unspecific binders tothe NiNTA matrix, a parallel purification was carried out side-by-side from M. smegmatis cells containing the plasmidwithout an inserted mtPUP gene. To rule out the possibilitythat differences in the lists of observed proteins are the result ofrun-to-run variance during the SCX and RP separations, weincorporated isobaric tagging for relative and absolutequantification (iTRAQ) of the control and specific eluatedigests into the workflow. The 2D liquid chromatographyserved to reduce sample complexity. Only two proteins(GroEL2, MSMEG_1583; CFP29, MSMEG_5830) were foundin negative control experiments as well, probably because ofthe presence of sequence segments with an ability to bind tothe NiNTA matrix with high affinity. The correspondingpeptide peaks were subtracted from the data set.
Cumulatively, the analysis led to the identification of 1661peptides that were observed only in the specific sample. Theycould be assigned to 243 putative pupylation targets, referredto as ‘candidate targets’ (Supplementary Table 1). For a proteinto be considered as a target, it had to be identified based on theconfident assignment of at least two strong collision-induceddissociation (CID) spectra. The analysis allowed a directassignment of pupylation sites to 32 mycobacterial targetproteins, referred to as ‘validated targets’ (Table I). For one ofthese, acyl carrier protein (MSMEG_4326), two distinctpupylation sites were observed. All pupylated peptides thatwere identified contained an internal lysine residue, which,due to the loss of the positive charge as a result of the bulkyPUP conjugation to the side chain e-amine, lost the ability tofunction as a tryptic cleavage site (Figure 2A and B). Thisobservation independently corroborated the evidence forcorrect pupylation assignment.
In parallel, the same samples were subjected to a comple-mentary 2D gel electrophoresis analysis (Figure 2C). Singlespots from the 2D gel were trypsinized and the resultingpeptides were analyzed by ESI MS/MS (Supplementary Table2). This approach led to the identification of 133 peptides thatcould be assigned to 13 validated pupylation substrates,covering a total of 17 confirmed pupylation sites (Table I).Three substrates were observed to be pupylated on more thana single lysine site: elongation factor Ts (MSMEG_2520, threesites, total number of lysines: 24), acyl carrier protein(MSMEG_4326, two sites, total number of lysines: 5) andalkylhydroperoxide reductase (MSMEG_4391, three sites, totalnumber of lysines: 11).
For further analysis, we pooled the identified pupylatedprotein sequences and sorted them with respect to the geneloci organization in M. smegmatis (see Table I for pooledvalidated targets and Supplementary Table 3A for pooledcandidate targets). The resulting list comprises 41 validatedmycobacterial pupylation targets with a total of 48 non-redundant lysine-mediated pupylation sites. Owing to thedirect demonstration of this type of lysine conjugation, these
Figure 1 SDS–PAGE gel analysis of mtPUP, expressed in Escherichia coli andMycobacterium smegmatis. All lanes contain protein eluates, after the NiNTApurification step. (A) mtPUP expression in E. coli. Lane 1, protein marker; andlane 2, polyhistidine-tagged PUP. (B) mtPUP expression in M. smegmatis. Lane1, protein marker; lane 2, polyhistidine-tagged mtPUP; and lane 3, control withvector without insertion. Additional control expression in M. smegmatis: lane 4,polyhistidine-tagged mtPUP(Q64A); lane 5, polyhistidine-tagged Rv3874(mtCFP-10).
Identification of mycobacterial pupylation targetsC Poulsen et al
2 Molecular Systems Biology 2010 & 2010 EMBO and Macmillan Publishers Limited
Table
IPro
tein
targ
ets
from
M.sm
egm
ati
sw
ith
one
or
more
confi
rmed
PU
Pm
odifi
cati
ons
Targ
et
no
Glo
bal
MS
2D
Gel
spots
Gen
elo
cus
(M.sm
egm
ati
s)
Det
ecte
dpupyla
tion
sequen
ces
Pupy-
late
d
lysi
nes
Gen
e
locu
s(M
.
tuber
culo
sis)
Pupyl-
atio
n
site
con-
serv
ed
Funct
ional
annota
tion
Func-
tional
cate
gori
-
zati
on
Gen
e
clust
erSpot
App.M
W
1M
SM
EG
_0024
HT
IFG
EV
VD
EESQ
KPupylG
GEV
VD
AIA
ST
PT
DR
K147
Rv0009
Pep
tidyl-
pro
lylci
s-tr
an
sis
om
eras
e2
21633
MSM
EG
_0643
YAD
GT
PIK
PupylG
GEA
SD
FT
YAIQ
RK
124
Extr
acel
lula
rso
lute
-bin
din
gpro
tein
,fa
mily
pro
tein
5
3632
MSM
EG
_0880
NVA
AG
AN
PLG
LK
PupylG
GER
K11
6Rv0440c
Y60-k
Da
chap
eronin
10
4278
MSM
EG
_1401
WV
KPupylG
GESV
EELM
EAV
DA
SIP
DPV
RK
188
Rv0685
Elo
nga
tion
fact
or
Tu2
5195
MSM
EG
_1484
LSEK
PupylG
GELG
IPQ
ISTG
DLFR
K23
Rv07
33
YA
den
yla
tekin
ase
7
61685
MSM
EG
_1522
GSR
KPupylG
GEST
PFA
AQ
LA
AEN
AA
RK
66
Rv3459
Y30
Sri
boso
mal
pro
tein
S11
2
711
02
MSM
EG
_2352
GV
GSA
EN
FK
PupylG
GEIV
EELA
DSLG
GAV
GA
SR
K21
0Rv3028c
Ele
ctro
ntr
ansf
erfl
avopro
tein
,al
pha
subunit
7
81553
MSM
EG
_2379
VD
KPupylG
GEAV
SEH
LA
TR
K31
3Rv2995c
3-I
sopro
pylm
alat
edeh
ydro
genas
e7
9171
2M
SM
EG
_2387
VVA
DV
LR
GR
KPupylG
GEVA
DG
VR
K380
Rv2988c
Y3-I
sopro
pylm
alat
edeh
ydra
tase
,la
rge
subunit
7I
1017
52
(21.7
)M
SM
EG
_2388
ND
PSFIL
NLSPFD
KPupylG
GEG
SV
LVA
GPD
FG
TG
SSR
K51
Rv2987c
Isopro
pylm
alat
eis
om
eras
esm
allsu
bunit
7
118
80
(29.3
)M
SM
EG
_2520
NA
LVEA
DG
DFD
KPupylG
GEAV
ELLR
K36
Y
AA
KPupylG
GETG
DT
TV
EQ
AIA
DLSA
KK
111
Rv2889c
YElo
nga
tion
fact
or
Ts
2
IGEK
PupylG
GELELR
K131
Y
213
MSM
EG
_2520
NA
LVEA
DG
DFD
KPupylG
GEAV
ELLR
IK
36
Rv2889c
YElo
nga
tion
fact
or
Ts
2
12
1601
MSM
EG
_2937
AT
TFY
DD
PD
VLA
KPupylG
GEV
SR
K275
Rv2606c
YPyri
doxal
bio
synth
esis
lyas
e7
13
511
0(4
2.5
)M
SM
EG
_305
5T
DIE
LPW
EQ
TN
KV
DD
LK
PupylG
GESA
IK
396
Rv1392c
YS-
aden
osy
lmet
hio
nin
esy
nth
etas
e7
14
1488
MSM
EG
_3081
LG
VSA
KPupylG
GEA
RK
172
Rv1423
YPuta
tive
unch
arac
teri
zed
pro
tein
9II
15
504
MSM
EG
_3084
VPIP
TG
SV
TD
LTA
ELA
KPupylG
GESA
SV
ED
INA
AM
KK
257
Rv1436
Gly
cera
ldeh
yde-
3-p
hosp
hat
edeh
ydro
genas
e7
16
706
MSM
EG
_310
2LEK
PupylG
GEIG
SD
EA
LA
LR
K21
9Rv1448
YTr
ansa
ldola
se7
17
1560
MSM
EG
_3205
GG
VD
VD
AV
VPK
PupylG
GEV
RPIV
DAVA
QR
K41
Rv1599
His
tidin
oldeh
ydro
genas
e7
18
1244
MSM
EG
_3461
FAPIN
SW
PD
NV
SLD
KPupylG
GEA
RK
149
Rv1908c
YC
atal
ase-
per
oxid
ase
20
19
539
MSM
EG
_3950
TLID
ASK
PupylG
GESA
QM
VV
VG
NR
K10
9Rv2026c
Univ
ersa
lst
ress
pro
tein
fam
ily
pro
tein
10
20
47
(10.6
)M
SM
EG
_4326
TV
GD
VVAY
IQK
PupylG
GELEEEN
PEA
AA
ALR
K79
Rv2244
YA
cylca
rrie
rpro
tein
1
III
20
284
MSM
EG
_4326
YG
VK
PupylG
GEIP
DED
LA
GLR
K58
Rv2244
YA
cylca
rrie
rpro
tein
1285
TV
GD
VVAY
IQK
PupylG
GELEEEN
PEA
AA
ALR
K79
2110
39
MSM
EG
_4327
VLED
EFV
TK
PupylG
GEW
DLPV
RK
53
Rv2245
Y3-o
xoac
yl-
(acy
l-ca
rrie
r-pro
tein
)sy
nth
ase
11
22
1387
MSM
EG
_4328
LSY
LQ
KPupylG
GEM
AT
VLG
RR
K84
Rv2246
3-o
xoac
yl-
(acy
l-ca
rrie
r-pro
tein
)sy
nth
ase
21
23
1523
MSM
EG
_4527
RA
QV
LVPELEK
PupylG
GER
K458
Rv2391
Ferr
edoxin
sulfi
tere
duct
ase
7
24
1866
MSM
EG
_4530
IAV
LN
KPupylG
GEG
RK
218
Rv2397
YSulf
ate
AB
Ctr
ansp
ort
er,AT
P-b
indin
gpro
tein
3
ALLT
IGD
QFPEY
DLT
AV
VG
GD
LSK
PupylG
GEV
DA
KK
25
Y
25
16
59
(21.5
)M
SM
EG
_4891
VD
AK
PupylG
GEQ
PD
DY
FT
RK
29
Rv2428
YA
lkylh
ydro
per
oxid
ere
duct
ase
0
VT
SK
PupylG
GED
YEG
KK
41
26
19
48
(42.5
)M
SM
EG
_4920
AA
AAW
KPupylG
GED
GV
FAD
EV
VPV
SIP
QR
K21
0Rv1323
YA
cety
l-C
oA
acet
ylt
ransf
eras
e1
27
1859
MSM
EG
_4954
KPupylG
GEN
GLR
RG
DAV
TG
AV
RK
320
Rv1297
YTr
ansc
ripti
on
term
inat
ion
fact
or
Rho
2IV
28
1165
(37.3
)M
SM
EG
_4956
KPupylG
GELT
AD
FPT
IALV
NSV
NPY
RK
151
Rv1295
YT
hre
onin
esy
nth
ase
7
29
161
4M
SM
EG
_510
4N
SLSG
AV
EV
VK
PupylG
GER
DG
TG
ITLN
EA
LH
AN
K299
Rv1201
cTe
trah
ydro
pic
olinat
esu
ccin
yla
se7
30
1291
MSM
EG
_5249
QPA
FQ
QYA
VA
DN
AQ
ALA
DG
FV
KPupylG
GER
DA
K362
Rv10
93
Ser
ine
hydro
xym
ethylt
ransf
eras
e7
3128
27
(13.1
)M
SM
EG
_5258
QA
IEG
FY
KPupylG
GEN
MTA
AT
TD
IKK
58
Ste
roid
del
tais
om
eras
e
32
15
63
(44.7
)M
SM
EG
_5335
WH
PD
IPA
AAT
VK
Pu
pylG
GQPG
SEFR
K36
Form
amid
ase
33
150
0M
SM
EG
_5431
TRTG
KPupylG
GEEG
ASR
K21
Rv10
15
YR
iboso
mal
pro
tein
L25,C
tc-f
orm
2
34
15
63
(30.9
)M
SM
EG
_5789
DFV
DA
FSK
PupylG
GELLSER
K67
Rv081
5c
YPuta
tive
thio
sulf
ate
sulf
urt
ransf
eras
e7
V554
MSM
EG
_5789
DFV
DA
FSK
PupylG
GELLSER
K67
Rv081
5c
YPuta
tive
thio
sulf
ate
sulf
urt
ransf
eras
e7
35
27,2
828
(8.9
)M
SM
EG
_5790
ALSSA
GN
GN
VT
VA
PTG
AG
IHEV
DV
KPupylG
GEVA
K88
Rv081
4c
YSse
Cpro
tein
7
67
MSM
EG
_5790
ALSSA
GN
GN
VT
VA
PTG
AG
IHEV
DV
KPupylG
GEVA
K88
Rv081
4c
YSse
Cpro
tein
7
36
1898
MSM
EG
_6008
ET
TLEK
PupylG
GEM
AA
LK
218
Rv3556c
YA
cety
l-C
oA
acet
ylt
ransf
eras
e1
Identification of mycobacterial pupylation targetsC Poulsen et al
& 2010 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2010 3
sequences can be considered to be unambiguously pupylatedunder the chosen experimental conditions. All candidatepupylation substrates revealed by 2D gel analysis were alsodetected in the ESI QqTOF analysis, except for MSMEG_4298,confirming the validity of the data (Supplementary Table 1).Remarkably, for four new validated targets we independentlyfound direct evidence for pupylation at identical sites by bothmethods: elongation factor Ts (MSMEG_2520), acyl carrierprotein (MSMEG_4326), a putative thiosulfate sulfurtransfer-ase (MSMEG_5789) and SseC (MSMEG_5790). All threepreviously identified pupylation targets (FabD, MIPS andSOD) were also observed in the complete list of potentialcandidate pupylation targets (Supplementary Table 1).
We also observed auto-pupylation at two sites, K31 and K61.However, as we were not able to distinguish between auto-pupylation of endogenous PUP from M. smegmatis andoverexpressed mtPUP, due to the high sequence similarity ofthe two proteins (92% identity), we cannot rule out thepossibility that some of the observed auto-pupylation may notreflect a physiological situation.
To analyze to what extent the M. smegmatis pupylome mayvary during cell growth and under stress conditions, wemonitored M. smegmatis culture growth in the presence ofestablished stress inducing agents and protocols (Darwin et al,2003), including hydrogen peroxide (to mimic oxidativestress), sodium nitrite (to mimic nitrosative stress) andepoxomicin (to inhibit proteasomal degradation; Supplemen-tary Figure 2). Under the latter two conditions, the M.smegmatis growth was completely inhibited, whereas appliedoxidative stress did not lead to any significant growth ratechanges. In addition, it is worth mentioning that PUPoverexpression on its own reduced M. smegmatis growthabout three-fold, when compared with wild-type M. smegma-tis cell cultures lacking a heterologous expression vector. In asubsequent 2D DIGE experiment (Supplementary Figure 3,Supplementary Table 4), we have identified and validated atotal of six spots observed in all three gels. These spots showsubstantial intensity variations under the three different testedconditions, indicating that the mycobacterial pupylome isdynamic.
Genetic and functional clustering of pupylationtargets
Although the genomes of several M. smegmatis strains havebeen sequenced (http://www.ncbi.nlm.nih.gov/nuccore/CP000480), no systematic functional annotation and categor-ization has yet been published. However, the vast majority(36/41) of our validated M. smegmatis targets with pupylationsites is mirrored by homologous proteins in M. tuberculosisH37Rv (Table I). Therefore, we have made use of availableanalyses of the related genome from the M. tuberculosis H37Rvstrain, in which the proteome was divided into ten differentfunctional categories (Camus et al, 2002). A total of 19 of thesetargets (47%) with homologs in M. tuberculosis H37Rv areinvolved in intermediary metabolism and respiration path-ways. Substantial numbers of targets are also found forcategories ‘lipid metabolism’ (six targets), ‘virulence, detox-ification, adaptation’ (four targets) and ‘information path-T
able
IC
onti
nued
Targ
et
no
Glo
bal
MS
2D
Gel
spots
Gen
elo
cus
(M.sm
egm
ati
s)
Det
ecte
dpupyla
tion
sequen
ces
Pupy-
late
d
lysi
nes
Gen
e
locu
s(M
.
tuber
culo
sis)
Pupyl-
atio
n
site
con-
serv
ed
Funct
ional
annota
tion
Func-
tional
cate
gori
-
zati
on
Gen
e
clust
erSpot
App.M
W
37
1769
MSM
EG
_6247
ELSA
DFD
LD
AFEH
VA
KPupylG
GEW
NSD
EER
IEM
WLR
K242
Rv370
1c
Puta
tive
unch
arac
teri
zed
pro
tein
10
38
805
MSM
EG
_6284
EQ
AT
WA
QK
PupylG
GEA
IAQ
EG
LTD
LA
EV
RK
247
Rv3720
YC
ycl
opro
pan
e-fa
tty-a
cyl-phosp
holipid
synth
ase
1V
I39
14
63
(46.3
)M
SM
EG
_6286
LG
ESK
PupylG
GEIA
SW
TD
PK
K339
Rv3722c
YA
spar
tate
tran
sam
inas
e10
40
24
37
(22.8
)M
SM
EG
_6427
HH
AT
YV
KPupylG
GEG
VN
DA
IAK
K38
Rv3846
Y[M
n]
super
oxid
edis
muta
se0
41165
MSM
EG
_651
8K
PupylG
GELQ
GSLA
EA
ASR
K30
Rv3863
Puta
tive
unch
arac
teri
zed
pro
tein
10
Pro
tein
targ
ets
wit
hpupyla
ted
sequen
ces
that
hav
ebee
nid
enti
fied
by
both
met
hods,
ESIQ
qTO
Fm
ass
spec
trom
etry
anal
ysi
san
d2D
gela
nal
ysi
s,ar
ehig
hligh
ted
ingr
ay.C
olu
mn
annota
tion:G
lobal
MS,p
epti
de
index
from
ESI-
QqTO
Fan
alysi
s(S
upple
men
tary
Table
1);
2D
gel,
pep
tide
index
from
2D
gel
elec
trophore
sis
(Supple
men
tary
Table
2);
App.
MW
(cal
cula
ted),
appar
ent
MW
ver
sus
calc
ula
ted
MW
(all
targ
ets
iden
tifi
edby
2D
gel
elec
trophore
sis
show
anap
par
ent
MW
that
exce
eds
the
calc
ula
ted
MW
of
the
sam
eta
rget
);G
ene
locu
s(M
.sm
egm
ati
s);D
etec
ted
pupyla
tion
pep
tides
,PU
PG
GE
site
sar
ehig
hligh
ted
by
super
scri
pt;
Pupyla
ted
lysi
nes
,se
quen
cere
sidue
num
ber
s;G
ene
locu
s(M
.tu
ber
culo
sis)
for
M.
smeg
mati
sge
nes
wit
hid
enti
fied
par
alogs
inM
.tu
ber
culo
sis;
Conse
rvat
ion
of
lysi
ne
pupyla
tion
site
,bas
edon
M.
smeg
mati
s/M
.tu
ber
culo
sis
sequen
ceal
ignm
ents
of
targ
ets
genes
(dat
anot
show
n);
Funct
ional
annota
tion
(M.
tuber
culo
sis)
;Funct
ional
cate
gori
zati
on
(M.
tuber
culo
sis)
,ca
tego
ryid
enti
fier
shav
ebee
nta
ken
from
(Cam
us
etal,
2002).
0,
vir
ule
nce
,det
oxifi
cati
on,ad
apta
tion;1,lipid
met
abolism
;2,i
nfo
rmat
ion
pat
hw
ays;
3,c
ellw
alla
nd
cell
pro
cess
es;7,in
term
edia
rym
etab
olism
and
resp
irat
ion;8,pro
tein
sofunknow
nfu
nct
ion;9,re
gula
tory
pro
tein
s;10
,co
nse
rved
hypoth
etic
pro
tein
s.Ta
rget
gene
clust
ers,
pai
ror
mult
iple
pupyla
tion
targ
ets
that
are
enco
ded
from
genes
wit
hnei
ghbori
ng
loci
,ge
ner
ally
found
injo
int
oper
ons,
are
indic
ated
I–V
I.
Identification of mycobacterial pupylation targetsC Poulsen et al
4 Molecular Systems Biology 2010 & 2010 EMBO and Macmillan Publishers Limited
ways’ (five targets). These categories are thought to be rich inpotential targets that could become useful for future drugdiscoveries (Zhang, 2005).
When expanding this analysis to the complete list ofM. smegmatis candidate targets we have identified as candi-date pupylation substrates (Supplementary Table 3B), we noticethat many of our observations for either overrepresentation or
underrepresentation of specific functional target categories inthe M. tuberculosis proteome serving as potential pupylationsubstrates are preserved (Figure 3). In our analysis, there is adistinct accumulation of potential pupylation substrate targetsinvolved virulence, detoxification and adaptation (category 0),lipid metabolism (category 1), information pathways (category 2)and intermediary metabolism and regulation (category 7), when
Y G V K I P D E D L A G L R
E
G
G
I V E E L A D S L G G A V G A S R
G
A B
VGSA
E
N
F
K
200 400 600 800 1000 1200
m/z (amu)
200 400 600 800 1000
m/z (amu)
100
G GE
80
60
40
20
0
Rel
ativ
e in
tens
ity (
%)
Rel
ativ
e in
tens
ity (
%) 100
60
20
114 115m/z (amu)
115.11
100
80
60
40
20
0
Rel
ativ
e in
tens
ity (
%)
Rel
ativ
e in
tens
ity (
%) 100
60
20
114 115m/z (amu)
115.11
C 150120
kDa pH
100
6075
50
85
25
40
30
20
15
3 4 5 6
Figure 2 Specific enrichment of pupylated peptide in polyhistidine affinity captured eluate. (A, B) Annotated CID spectra that contributed to the identification of (A)MSMEG_2352 and (B) MSMEG_4326 as a pupylation targets. The CID spectrum of (A) was derived by fragmentation of a quadruple-charged precursor ion observed atm/z [766.91þ 4H]4þ and was matched to the branched peptide GVGSAENFK(QGG)IVEELADSLGGAVGASR carrying iTRAQ labels at the N-termini of both its mainand branched chain. Please note the absence of a detectable iTRAQ114 signature mass peak (inset), indicating that this peptide was exclusively contributed by thepupylated iTRAQ 115-labeled sample. Also note the double charged nature of branched peptide fragments due to the retention of an additional charge by the primaryamine present within the N-terminal glycine of the GGQ pupylation stub. The CID spectrum of (B) was derived by fragmentation of a triple charged precursor ionobserved at m/z [693.37 þ 3H]3þ and was matched to the branched peptide YGVK(QGG)IPDEDLAGLR, carrying iTRAQ labels at the N-termini of both its main andbranched chain. For information on additional peptides see Table I. (C) Complementary 2D gel analysis. Pupylated substrates were separated on a 2D gel and spots arenumbered, which are subjected to tandem MS/MS analyses. The numbering of the spots corresponds to the numbering used in the tables. The pH range of the firstdimension and apparent molecular weight of second dimension are denoted.
Identification of mycobacterial pupylation targetsC Poulsen et al
& 2010 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2010 5
compared with the overall presentation of targets from thesecategories within the entire proteome. In contrast, the number ofpotential pupylation substrate targets involved in cell wall andcell processes (category 3) is small, compared with its overallpresentation within the complete proteome. Finally, we haveobserved no evidence for any pupylation substrates that areidentified as targets in functional categories 4, 5 and 6.
We have also carried out an analysis of the pupylated targetsin terms of gene locus organization. Strikingly, a substantialfraction of the identified targets with identified conjugationsites (13/41) is located in six gene clusters, I–VI (Table I),containing at least two genes encoded within one operon or inclose proximity. This observation suggests that protein targetpupylation could be correlated with functional linkages(Dandekar et al, 1998; Overbeek et al, 1999; Pellegrini et al,1999). Noticeably, gene locus clustering of potential pupyla-tion targets becomes even more obvious when the completelist of candidate pupylation targets is taken into account(Supplementary Table 3A).
Outlook
The availability of the complete genome and the correspond-ing proteome of M. tuberculosis has revolutionized funda-mental research and generated new approaches to studydisease mechanisms (Cole et al, 1998; Mattow et al, 2001).However, little is known about posttranslational modificationsof targets from the M. tuberculosis proteome and relatedmycobacteria, except for phosphorylation (Greenstein et al,2005; Wehenkel et al, 2008). In this contribution, we haveshown for M. smegmatis that a substantial number of proteinsmay serve as targets for covalent modification by PUPconjugation, ultimately leading to alteration of their functionalstatus and perhaps their fate for controlled proteasome-mediated degradation.
An important task for the future is to experimentally map theavailable evidence for pupylation of M. smegmatis targets ontothe proteome of pathogenic M. tuberculosis strains and otherpathogenic mycobacteria. Furthermore, it will be of specificinterest to determine to what extent pupylation of targets mayvary during different stages of the mycobacterial life cycle andwithin the host environment; this will require studies inappropriate in vivo models. Future studies exploring thesignificance of pupylation for fundamental biological pro-cesses in mycobacteria will be needed to elucidate aspects ofpupylation biology that may find applications in ongoingefforts to overcome tuberculosis and other mycobacterialdiseases.
Materials and methods
M. smegmatis growth under different experimentalconditions
Bacterial growth was monitored by measuring the optical densities at600 nm (OD600) as a function of time at a wavelength of 600 nm, intriplicates. Cultures were prepared in identical triplicates for each timepoint, thereby ensuring that the growth of bacteria was not disturbeduntil the measurement time. Each cell density with an OD600 valueexceeding 1.5 was diluted to allow density measurements within thelinear range of the detector. Specific stress conditions were generated,by adding 2 mM H2O2, 3 mM NaNO2 (pH 5.5) or 20 mM epoxomicin(Enzo Life Sciences) to the growth medium.
Expression and purification of mtPUP
The ORF of PrcB, PrcA and PUP (Rv2109c, Rv2110c and Rv2111c) wasamplified from the H37Rv genomic DNA by PCR, using the forwardprimer 50-GAGCCATGGCGCAAGAGCAGACCAA-30 and the reverseprimer 50-GAGAAGCTTCAGCCCGACGATTCGCC-30. For cloning of themtPUP(Q64A) variant, the forward primer 50-GAGCCATGGCGCAAGAGCAGACCAA-30 and reverse primer 50-GCGGGATCCTTATCACGCTCCGCCCTTTTGGAC-30 were used. The PCR-introduced restriction sites,
Figure 3 Functional analysis of pupylation targets. Relative distribution of validated PUP targets (gray bars, for details see Table I) and candidate PUP targets (blackbars, for details see Supplementary Tables 3A and B) with M. tuberculosis homologs for different functional categories (Camus et al, 2002). The functional distribution ofthe M. tuberculosis proteome is shown for comparison (white bars).
Identification of mycobacterial pupylation targetsC Poulsen et al
6 Molecular Systems Biology 2010 & 2010 EMBO and Macmillan Publishers Limited
NcoI and HindIII were used to clone the PCR product into a modifiedpSD26 vector in M. smegmatis (Daugelat et al, 2003). For heterologousexpression in E. coli, the PCR product was cloned into a pETM-11vector (Dummler et al, 2005). We used DNA sequencing to verify thatboth constructs contained the correct insert. For mtPUP expressionin M. smegmatis, we used a previously established protocol (Ma et al,2006). The pMyNT construct and empty pMyNT vector weretransformed into M. smegmatis MC2 155 by electroporation andgrown on 7H10 agar plates supplemented with 0.5% glycerol, 0.05%Tween 80, ADS (5 g/l BSA, 2 g/l D-dextrose and 0.8 g/l NaCl). In bothplates and medium, 50mg/ml hygromycin was added as a selectionmarker. Overnight pre-cultures in 7H9 broth supplemented with 0.5%glycerol, 0.05% Tween 80 and ADS were added to 2 l of expressionmedium (7H9 broth supplemented with 0.2% glycerol, 0.05% Tween80 and 2 g/l glucose). Induction was started, by adding 30 mMacetamide. Depending on the experimental conditions, induction wascarried out at a different cell density as specified in the result section.For pupylome analysis the cells were collected by centrifugation andresuspended in lysis buffer containing 50 mM Tris–HCl (pH 8.0), 8 Murea, protease inhibitors cocktail (Serva #39106) containing AEBSF-HCl, aprotinin, E64, and leupeptin, and 40 mM imidazole. Lysis wascarried out by sonication; after centrifugation, the soluble fraction wasfiltered through a 0.22-mm filter and passed over a 200-ml NiNTA resincolumn. Affinity-bound proteins were eluted with elution buffercontaining 300 mM imidazole and 6 M urea.
For mtPUP expression in E. coli, the pETM-11 construct wastransformed into BL21 DE3 RIL and plated on agar plates containing50mg/ml kanamycin and 37mg/ml chloramphenicol. Pre-cultures wereadded to LB medium and 1 mM IPTG was used to induce theexpression at an OD600 value of 0.8. The induced cells were leftovernight at 201C before collecting. The cells were resuspended inlysis/washing buffer: 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 1 Murea and protease inhibitors. The protein was purified as described,but in the absence of denaturing agents. Eluted protein was exchangedto 50 mM Tris–HCl (pH 8.0) and 100 mM NaCl for TEV proteasecleavage. Uncleaved His-tagged protein, TEV and other contaminantswere removed by passing the sample through NiNTA resin.
For immunoblot analysis, purified proteins were separated on SDS–PAGE. The bands were electrotransferred on to a polyvinylidenedifluoride membrane, which was treated with blocking solution(Sigma) in PBST buffer (25 mM phosphate buffer (pH 7.4), 125 mMNaCl and 0.05% Tween-20). Polyhistidine-tagged proteins weredetected with a horseradish peroxidase (HRP)-linked anti-penta-hisantibody (Qiagen), using the Enhanced Chemiluminescence (ECL)detection kit (GE Healthcare).
ESI QqTOF mass spectrometry analysis foridentification of PUP-conjugated targets
Protein-containing fractions were denatured in the presence of 6 Murea, 20 mM NH4HCO3 (pH 8.0), followed by reduction with 1 mMTris-(2-carboxyethyl)-phosphine for 30 min at 601C and alkylationwith 2.5 mM 4-vinylpyridine for 1 h at room temperature in the dark.Samples were diluted four-fold to ensure that the concentration of ureadid not exceed 1.5 M. Tryptic digestion was initiated by the addition of1% (wt/wt) of side chain-modified, TPCK-treated porcine trypsin andallowed to proceed at 371C for 6 h.
Individual iTRAQ labeling reagents (Applied Biosystems, FosterCity, CA, USA) were reconstituted in ethanol, added to peptidemixtures derived from the tryptic digestion of NiNTA eluate fractions(control, iTRAQ 114; sample, TRAQ 115) and incubated at roomtemperature in the dark for 3 h.
The column effluent was coupled directly via a fused silica capillarytransfer line to a QSTAR XL hybrid quadrupole/time-of-flight tandemmass spectrometer (Applied Biosystems; MDS Sciex, Concord, ON,Canada) equipped with a MicroIonSpray source. The progress of eachLC/MS run was monitored by recording the total ion current (TIC) as afunction of time for ions in the m/z range 300–1800. At 5-s intervalsthrough the gradient, a mass spectrum was acquired for 1 s, followedby one CID acquisition of 4 s each on ions selected by presetparameters of the information-dependent acquisition (IDA) method,using nitrogen as the collision gas. Singly charged ions were excluded
from CID selection. The collision energy was adjusted automaticallyfor each CID spectrum using an empirically optimized formula thatconsiders the charge state and m/z value of the precursor ion.
Strong cation exchange (SCX) chromatography was used to achievepeptide fractionation of the complex digest mixture. Samples digestedwith trypsin were adjusted to 25% acetonitrile and acidified (pH 3.0)by 20-fold dilution in 25% acetonitrile and 20 mM KH2PO4 (pH 3.0).High performance liquid chromatography was carried out using theUltimate System (Dionex, Sunnyvale, CA, USA), equipped with amicroflow calibration cartridge, a Valco injection port and a 180 nl UVcell. Separation was achieved on a self-packed 0.5 mm� 110 mm LunaSCX column (Phenomenex, Torrance, CA, USA), at a flow rate of 18ml/min and with a salt gradient of 0–400 mM NH4Cl, in 25% acetonitrileand 20 mM KH2PO4 (pH 3.0). Fractions eluted from the SCX columnwere desalted with C18 Empore (3 M, Minneapolis, MN, USA) stop andgo extraction tips (Rappsilber et al, 2003), and subsequently subjectedto a nano-flow RP-HPLC using the Ultimate LC system (Dionex)equipped with a nano-flow calibration cartridge at a flow rate of250 nl/min. Peptides were separated on a 75-mm ID self-packedcolumn containing a Proteo C12 reverse-phase matrix (Phenomenex),using a 100-min gradient of 2–34% acetonitrile in water, with 0.1%(w/v) formic acid as ion-pairing agent.
The analysis of the iTRAQ data was carried out with the softwareprogram ProteinPilot (Applied Biosystems; MDS Sciex). Raw iTRAQratios were corrected for impurity levels of individual reagent lots,determined by the manufacturer. Alternatively, peak lists for databasesearching were created using Mascot Distiller (Version 1; Ma-trixScience, London, UK), and searches were performed usingdesignated MS/MS data interpretation algorithms within Mascot(Version 2.2; MatrixScience). Modifications considered were theattachment of GGQ or GGE motifs to lysine residues, assuming acondensation reaction that proceeds with the concomitant loss of awater molecule and possible deamidation. In a negative control dataanalysis, a similar modification was assumed. For ProteinPilotsearches, the algorithm assumes by default all possible unexpectedcleavages and many of the more common modifications. Searchesfurther considered up to two missed cleavages and charge statesranging from þ 2 to þ 4. In the few instances in which confidence valuesfor assigned pupylation sites were not meeting or exceeding 95%confidence levels, the lower confidence, and thus higher assumed errorrate, was partially offset by the presence of corroborating missedcleavages at the pupylated lysine residues and manual inspectionof CID spectra. In particular, the latter approach led in a subset of spectrato the detection of candidate pupylation fragments that increased theconfidence of assignments but were not considered by the algorithms forthe calculation of scores. The experimental data have been submitted tothe PRIDE data base (accession number: 11999).
For the analysis of 2D gel spots, these were manually isolated fromthe gel, washed three times with 10 mM ammonium bicarbonate buffer(pH 7.8) and in-gel digested with trypsin (Promega, Mannheim,Germany) at 371C overnight. For nano-HPLC/ESI-MS/MS analyses,tryptic peptides were extracted twice from gel pieces with 50% ACN in0.1% trifluoroacetic acid. The MS/MS analyses were performed on ahigh-capacity ion trap system (HCT ultra, Bruker Daltonics), inconjunction with an online RP nano-HPLC system (Dionex U3000,Dionex LC Packings, Idstein, Germany).The mass spectrometer wasoperated in the sensitive mode with the following parameters:capillary voltage, 1400 V; end plate offset, 500 V; dry gas, 8.0 l/min;dry temperature, 1601C; aimed ion charge control, 150 000; maximalfill-time, 500 ms. The nano-ESI source (Bruker Daltonics) wasequipped with distal coated silica tips (FS360–20–10-D; New Objec-tive). The MS spectra were recorded from the sum of seven individualscans, ranging from m/z 300 to m/z 1500, with a scanning speed of8100 (m/z)/s. The three most intense, multiple-charged peptide ionsdetected within the MS spectra were selected in a data-dependentmanner (HCTultra, Esquire Control) to subsequently perform MS/MS.To generate fragment ions, low-energy CID was performed on previouslyisolated peptide ions by applying a fragmentation amplitude of 0.6 V.Generally, MS/MS spectra were the sum of four scans ranging from m/z100 to m/z 2200, at a scan rate of 26 000 (m/z)/s. Exclusion limits wereautomatically placed on previously selected mass-to-charge ratios for1.2 min. The ion trap instrument was externally calibrated withcommercially available standard compounds.
Identification of mycobacterial pupylation targetsC Poulsen et al
& 2010 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2010 7
For protein identification, raw MS/MS data were searched usingthe Mascot algorithm (Matrix Science, v.2.2.0) against the NCBInrdatabase (v.23.01.2009, 7 699 833 protein entries), restricted tobacteria, with the following search parameters: fixed modification oncysteines, due to propionamidation; variable modifications due tomass shift of 243.085521 Da on pupylated lysines, resulting from atryptic fragment of mtPUP (sequence GGE) attached via an isopeptidebond (Pearce et al, 2008), and methionine oxidation. In addition, twomaximal missed cleavage sites, in case of incomplete trypsinhydrolysis and a mass tolerances of ±1.2 Da for parent ions and±0.4 Da fragment ions, were considered for database searches. Thereliability of protein identification was verified manually; a mascotscore 460 was required to consider protein identification assignificant. The experimental data have been submitted to the PRIDEdata base (accession number: 12078).
2D gel electrophoresis
For 2D gel electrophoresis, protein eluate from the NiNTA preparationwas treated three times with a buffer (30 mM Tris, 7 M urea, 2 Mthiourea and 4% CHAPS (pH 8.5)) compatible with isoelectric focusingusing Microcons (Millipore, Schwalbach, Germany) with a 3-kDacut-off. For IEF, a 20-mg sample was prepared by adding 0.7ml DTT(1.08 g/ml; Bio-Rad) and 0.7ml Servalyt 2–4 (Serva). Carrier ampho-lyte-based IEF was performed in a self-made IEF chamber, using tubegels (20 cm� 1.5 mm) as described elsewhere (Sitek et al, 2006).Briefly, after running a 21-h voltage gradient, the ejected tube gels wereincubated in equilibration buffer (125 mM Tris, 40% (w/v) glycerol,3% (w/v) SDS and 65 mM DTT (pH 6.8)) for 10 min. The seconddimension was performed in a Desaphor VA 300 system (Sarstedt,Numbrecht, Germany) using polyacrylamide gels (15.2% totalacrylamide and 1.3% bisacrylamide) as described elsewhere (Siteket al, 2006). Therefore, the IEF tube gels were placed onto thepolyacrylamide gels (20 cm� 30 cm� 1.5 mm) and fixed using 1.0%(wt/vol) agarose containing 0.01% (wt/vol) bromophenol blue dye(Riedel deHaen, Seelze, Germany). Silver staining was performedusing an MS-compatible protocol (Blum et al, 1986).
To analyze the differential expression of proteins under differentgrowth conditions, we have used the 2D DIGE method (Unlu et al,1997). Before electrophoresis, the protein samples were labeled withthree different fluorescence CyDyes (GE Healthcare). The fluorescencedye stock solutions (1 nmol/ml) were diluted with anhydrous DMF(400 pmol/ml) and 8 pmol dye was added per microgram protein. Thesamples were mixed thoroughly, cleared by centrifugation and thelabeling reaction was carried out in the dark by incubating on ice for0.5 h. The reaction was stopped by adding 1ml of 10 mM L-lysine per8 pmol dye.
Multiple PUP sequence alignment
All PUP homologs of mtPUP (Rv2111) were obtained in a global BLASTsearch. All unique sequences below an E-value of 1.0 were selected forthe alignment. The alignment was carried out with ClustalX (2.0.9;Larkin et al, 2007). The secondary structure prediction was carried outusing the computational server of Pred and PROF (Ouali and King,2000).
Functional annotation and gene clustering
The identifiers of M. smegmatis and M. tuberculosis genes havebeen taken from the Comprehensive Microbial Resources(http://cmr.jcvi.org). The identification of genes with paralogs inM. smegmatis and M. tuberculosis were carried out with the ArtemisComparison Tool (Carver et al, 2005). Functional annotation andcategorization (M. tuberculosis) has been retrieved from TubercuList(http://tuberculist.epfl.ch). Category identifiers were defined by(Camus et al, 2002). Pupylation targets were grouped in one genecluster according to the following criteria: (a) genetic vicinity,for example, consecutive location and/or encoded in the same operon;and (b) conservation of gene order in M. smegmatis andM. tuberculosis.
Supplementary information
Supplementary information is available at the Molecular SystemsBiology website (www.nature.com/msb).
AcknowledgementsYA thanks DAAD (German Academic Exchange Service) for a long-term doctoral fellowship. We thank Morlin Milewski for technicalassistance. This study was supported by grants from the CanadianInstitutes of Health Research (CIHR; MOP-74734 (GSU)), from BMBF(Pathogenomics-Plus, PTJ-BIO 0313801L) and from the EuropeanCommission Framework VII (NATT, 222965) to MW.
Conflict of interestThe authors declare that they have no conflict of interest.
References
Blum H, Beier H, Gross HJ (1986) Improved silver staining of plantproteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99
Burns KE, Liu WT, Boshoff HI, Dorrestein PC, Barry III CE (2009)Proteasomal protein degradation in Mycobacteria is dependentupon a prokaryotic ubiquitin-like protein. J Biol Chem 284:3069–3075
Camus JC, Pryor MJ, Medigue C, Cole ST (2002) Re-annotation of thegenome sequence of Mycobacterium tuberculosis H37Rv.Microbiology 148: 2967–2973
Carver TJ, Rutherford KM, Berriman M, Rajandream MA, Barrell BG,Parkhill J (2005) ACT: the artemis comparison tool. Bioinformatics21: 3422–3423
Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, GordonSV, Eiglmeier K, Gas S, Barry III CE, Tekaia F, Badcock K, Basham D,Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T,Gentles S et al (1998) Deciphering the biology of Mycobacteriumtuberculosis from the complete genome sequence. Nature 393:537–544
Crosas B, Hanna J, Kirkpatrick DS, Zhang DP, Tone Y, Hathaway NA,Buecker C, Leggett DS, Schmidt M, King RW, Gygi SP, Finley D(2006) Ubiquitin chains are remodeled at the proteasome byopposing ubiquitin ligase and deubiquitinating activities. Cell 127:1401–1413
Dandekar T, Snel B, Huynen M, Bork P (1998) Conservation of geneorder: a fingerprint of proteins that physically interact. TrendsBiochem Sci 23: 324–328
Darwin KH, Ehrt S, Gutierrez-Ramos JC, Weich N, Nathan CF (2003)The proteasome of Mycobacterium tuberculosis is required forresistance to nitric oxide. Science 302: 1963–1966
Daugelat S, Kowall J, Mattow J, Bumann D, Winter R, Hurwitz R,Kaufmann SH (2003) The RD1 proteins of Mycobacteriumtuberculosis: expression in Mycobacterium smegmatis andbiochemical characterization. Microbes Infect 5: 1082–1095
Dummler A, Lawrence AM, de Marco A (2005) Simplified screening forthe detection of soluble fusion constructs expressed in E. coli usinga modular set of vectors. Microb Cell Fact 4: 34
Grabbe C, Dikic I (2009) Functional roles of ubiquitin-like domain(ULD) and ubiquitin-binding domain (UBD) containing proteins.Chem Rev 109: 1481–1494
Greenstein AE, Grundner C, Echols N, Gay LM, Lombana TN,Miecskowski CA, Pullen KE, Sung PY, Alber T (2005) Structure/function studies of Ser/Thr and Tyr protein phosphorylation inMycobacterium tuberculosis. J Mol Microbiol Biotechnol 9: 167–181
Hatfull GF, Cresawn SG, Hendrix RW (2008) Comparative genomics ofthe mycobacteriophages: insights into bacteriophage evolution. ResMicrobiol 159: 332–339
Identification of mycobacterial pupylation targetsC Poulsen et al
8 Molecular Systems Biology 2010 & 2010 EMBO and Macmillan Publishers Limited
Hershko A, Ciechanover A (1986) The ubiquitin pathway for thedegradation of intracellular proteins. Prog Nucleic Acid Res Mol Biol33: 19–56, 301
Hershko A, Ciechanover A (1998) The ubiquitin system. Annu RevBiochem 67: 425–479
Iyer LM, Burroughs AM, Aravind L (2008) Unraveling the biochemistryand provenance of pupylation: a prokaryotic analog ofubiquitination. Biol Direct 3: 45
Kremer L, Dover LG, Carrere S, Nampoothiri KM, Lesjean S, Brown AK,Brennan PJ, Minnikin DE, Locht C, Besra GS (2002) Mycolic acidbiosynthesis and enzymic characterization of the beta-ketoacyl-ACPsynthase A-condensing enzyme from Mycobacterium tuberculosis.Biochem J 364: 423–430
Kusunose E, Ichihara K, Noda Y, Kusunose M (1976) Superoxidedismutase from Mycobacterium tuberculosis. J Biochem 80:1343–1352
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA,McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, ThompsonJD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version2.0. Bioinformatics 23: 2947–2948
Ma Q, Zhao X, Eddine AN, Geerlof A, Li X, Cronan JE, Kaufmann SH,Wilmanns M (2006) The Mycobacterium tuberculosis LipB enzymefunctions as a cysteine/lysine dyad acyltransferase. Proc Natl AcadSci USA 103: 8662–8667
Mattow J, Jungblut PR, Schaible UE, Mollenkopf HJ, Lamer S, Zimny-Arndt U, Hagens K, Muller EC, Kaufmann SH (2001) Identificationof proteins from Mycobacterium tuberculosis missing in attenuatedMycobacterium bovis BCG strains. Electrophoresis 22: 2936–2946
Ouali M, King RD (2000) Cascaded multiple classifiers for secondarystructure prediction. Protein Sci 9: 1162–1176
Overbeek R, Fonstein M, D’Souza M, Pusch GD, Maltsev N (1999) Theuse of gene clusters to infer functional coupling. Proc Natl Acad SciUSA 96: 2896–2901
Pearce MJ, Mintseris J, Ferreyra J, Gygi SP, Darwin KH (2008)Ubiquitin-like protein involved in the proteasome pathway ofMycobacterium tuberculosis. Science 322: 1104–1107
Pellegrini M, Marcotte EM, Yeates TO (1999) A fast algorithm forgenome-wide analysis of proteins with repeated sequences.Proteins 35: 440–446
Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips formatrix-assisted laser desorption/ionization, nanoelectrospray, andLC/MS sample pretreatment in proteomics. Anal Chem 75: 663–670
Sitek B, Potthoff S, Schulenborg T, Stegbauer J, Vinke T, Rump LC,Meyer HE, Vonend O, Stuhler K (2006) Novel approaches to analyseglomerular proteins from smallest scale murine and humansamples using DIGE saturation labelling. Proteomics 6: 4337–4345
Stieglitz KA, Yang H, Roberts MF, Stec B (2005) Reaching formechanistic consensus across life kingdoms: structure andinsights into catalysis of the myo-inositol-1-phosphate synthase(mIPS) from Archaeoglobus fulgidus. Biochemistry 44: 213–224
Striebel F, Imkamp F, Sutter M, Steiner M, Mamedov A, Weber-Ban E(2009) Bacterial ubiquitin-like modifier Pup is deamidated andconjugated to substrates by distinct but homologous enzymes.Nat Struct Mol Biol 16: 647–651
Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis:a single gel method for detecting changes in protein extracts.Electrophoresis 18: 2071–2077
Wehenkel A, Bellinzoni M, Grana M, Duran R, Villarino A, FernandezP, Andre-Leroux G, England P, Takiff H, Cervenansky C, Cole ST,Alzari PM (2008) Mycobacterial Ser/Thr protein kinases andphosphatases: physiological roles and therapeutic potential.Biochim Biophys Acta 1784: 193–202
Zhang Y (2005) The magic bullets and tuberculosis drug targets. AnnuRev Pharmacol Toxicol 45: 529–564
Molecular Systems Biology is an open-access journalpublished by European Molecular Biology Organiza-
tion and Nature Publishing Group. This work is licensed under aCreative Commons Attribution-Noncommercial-No DerivativeWorks 3.0 Unported License.
Identification of mycobacterial pupylation targetsC Poulsen et al
& 2010 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2010 9