Comparison of transcriptional and translational changes caused by long-term menadione exposure in Aspergillus nidulans
Full text
1
2Compa ison o ansc ip ional and ansla ional changes caused by long- e m
3menadione exposu e in Aspe gillus nidulans
4Tünde Pusz ahelyi
a,
⇑
, É a Klemen
b
, Emilia Szajli
b
, Józse Klem
c,d
, Má on Miskei
e,
,
5Zsol Ka ányi
g
, Tamás Em i
a
, Szil ia Ko ács
a
, Gyula O osz
d
, Ko nél L. Ko ács
d
, Ka alin F. Medzih adszky
b,h
,
6Rol A. P ade
i
, Is án Pócsi
a,
⇑⇑
7
a
Depa men o Mic obial Bio echnology and Cell Biology, Facul y o Science and Technology, Uni e si y o Deb ecen, Egye em é 1, H-4032 Deb ecen, Hunga y
8
b
P o eomics Resea ch G oup, Ins i u e o Biochemis y, Biological Resea ch Cen e , Hunga ian Academy o Sciences, Temes á i k . 62, H-6726 Szeged, Hunga y
9
c
Ins i u e o Biophysics, Biological Resea ch Cen e , Hunga ian Academy o Sciences, Temes á i k . 62, H-6726 Szeged, Hunga y
10
d
Depa men o Bio echnology, Uni e si y o Szeged, Közép aso 52, H-6726 Szeged, Hunga y
11
e
Depa men o Ho icul u al Sciences and Plan Bio echnology, Facul y o Ag icul u e, Cen e o Ag icul u al Sciences, Uni e si y o Deb ecen, Egye em é 1, H-4032
12 Deb ecen, Hunga y
13
Mycology G oup o he Hunga ian Academy o Sciences, Ins i u e o Plan P o ec ion, Szen Is án Uni e si y, Pá e Ká oly u ca. 1, H-2100 Gödöll}
o, Hunga y
14
g
Depa men o Medicine, Facul y o Medicine, Uni e si y o Deb ecen, P.O. Box 19, H-4012 Deb ecen, Hunga y
15
h
Depa men o Pha maceu ical Chemis y, Uni e si y o Cali o nia San F ancisco, San F ancisco, CA 94158, USA
16
i
Depa men o Biochemis y and Molecula Biology, Oklahoma S a e Uni e si y, 348E Noble Resea ch Cen e , S illwa e , OK 74078, USA
17
19
a icle in o
20 A icle his o y:
21 Recei ed 22 Feb ua y 2010
22 Accep ed 19 Augus 2010
23 A ailable online xxxx
24 Keywo ds:
25 Fungi
26 Aspe gillus (Eme icella)nidulans
27 Oxida i e s ess
28 Menadione
29 P o eomics
30 Genomics
31
32
abs ac
33
Unde long- e m oxida i e s ess caused by menadione sodium bisulfi e, genome-wide ansc ip ional
34
and p o eome-wide ansla ional changes we e compa ed in Aspe gillus nidulans ege a i e cells. The
35
compa ison o p o eomic and DNA mic oa ay exp ession da a demons a ed ha global gene exp ession
36
changes eco ded wi h ei he flip-flop o dend ime cDNA labeling echniques suppo ed p o eome
37
changes mode a ely wi h 40% and 34% coincidence coe ficien s, espec i ely. Enzyme le els in he glyco-
38
ly ic pa hway we e al e na ing, which was a di ec consequence o fluc ua ing gene exp ession pa e ns.
39
Su p isingly, enzymes in he i amin B2 and B6 biosyn he ic pa hways we e ep essed concomi an ly
40
wi h he ep ession o some p o ein olding chape ones and nuclea anspo elemen s. Unde long- e m
41
oxida i e s ess, he pe oxide-de oxi ying pe oxi edoxins and cy och ome c pe oxidase we e eplaced by
42
hio edoxin educ ase, a ni o educ ase and a fla ohemoglobin, and p o ein deg ada ion became p edom-
43
inan o elimina e damaged p o eins.
44
Ó2010 Published by Else ie Inc.
45
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1. In oduc ion
49
Inae obico ganisms eac i eoxygenspecies (ROS)a egene a ed
50
con inuously as side p oduc s o espi a ion (Li e al., 2009). ROS in-
51
clude hyd ogen pe oxide (H
2
O
2
), supe oxide anion (O
2
) and hyd o-
52
xyl adicals (HO
). In addi ion o hei impo an signaling unc ions
53
in di e se cellula p ocesses (La a-O íz e al., 2003; Cano-Domin-
54
guez e al., 2008), ROS a e also cy o oxic in p oka yo ic and euka y-
55
o ic o ganisms. No su p isingly, significan e o s a e made by he
56
O
2
-exposed cells o elimina e ha m ul ROS h ough a wide a ay o
57
bo h enzyma ic and non-enzyma ic p ocesses (Pócsi e al., 2004; Li
58
e al., 2009). Highe concen a ions o ROS ha may o igina e om
59
exogenous sou ces o due o in acellula enzyme ac i i ies may
60
cause aging and e en ini ia e apop o ic cell dea h (Pe one e al.,
61
2008; Scheckhube e al., 2009). ROS gene a ed a low concen a-
62
ions can igge an adap i e s ess esponse ha makes he cells
63
esis an o le hal concen a ions o hese oxic oxygen de i a i es
64
(Collinson and Dawes, 1992; Jamieson, 1992; Li e al., 2008a).
65
Gene exp ession and p o eome su eys ha e iden ified nume -
66
ous genes and gene p oduc s induced o ep essed in esponse o
67
oxidan s in yeas s and filamen ous ungi (Godon e al., 1998; Gasch
68
e al., 2000; Chen e al., 2003, 2008; Kim e al., 2006, 2007a).
69
Applica ions o ROS gene a ing agen s, employed a suble hal doses
70
in Aspe gillus nidulans (Pócsi e al., 2005) and Saccha omyces ce e i-
71
siae (Gasch e al., 2000; Tho pe e al., 2004), e ealed significan
72
di e ences in gene exp ession depending on ea u ed chemical,
73
he concen a ions o he applied agen s and he p oduced ROS.
1087-1845/$ - see on ma e Ó2010 Published by Else ie Inc.
doi:10.1016/j. gb.2010.08.006
Abb e ia ions: GSH, glu a hione; GSSG, glu a hione disulfide; ROS, eac i e
oxygen species; MSB, menadione sodium bisulfi e.
⇑
Co esponding au ho . Add ess: Depa men o Mic obial Bio echnology and
Cell Biology, Facul y o Science and Technology, Uni e si y o Deb ecen, P.O. Box 63,
H-4010 Deb ecen, Hunga y.
⇑⇑
Co esponding au ho . Add ess: Depa men o Mic obial Bio echnology and
Cell Biology, Facul y o Science and Technology, Uni e si y o Deb ecen, P.O. Box 63,
H-4010 Deb ecen, Hunga y.
E-mail add esses: [email p o ec ed] (T. Pusz ahelyi), [email p o ec ed]
(I. Pócsi).
Fungal Gene ics and Biology xxx (2010) xxx–xxx
Con en s lis s a ailable a ScienceDi ec
Fungal Gene ics and Biology
jou nal homepage: www.else ie .com/loca e/y gbi
YFGBI 2267 No. o Pages 12, Model 5G
30 Augus 2010
Please ci e his a icle in p ess as: Pusz ahelyi, T., e al. Compa ison o ansc ip ional and ansla ional changes caused by long- e m menadione exposu e
in Aspe gillus nidulans. Fungal Gene . Biol. (2010), doi:10.1016/j. gb.2010.08.006
74
Pócsi e al. (2005) ca ied ou genome-le el gene exp ession da a
75
analysis on he oxida i e s ess esponse o A. nidulans, and hey
76
ound ha 2499 o he 3533 unique PCR-amplified gene p obes
77
p in ed on an EST-based DNA mic oa ay we e a ec ed by a leas
78
one o he oxida i e s ess gene a ing agen s diamide, H
2
O
2
and
79
menadione sodium bisulfi e (MSB).
80
Unde he expe imen al condi ions used by Pócsi e al. (2005),
81
diamide, which is a hiol oxidizing compound, caused a quick
82
change in he glu a hione/glu a hione disulfide (GSH/GSSG) edox
83
s a us o he cells wi hou influencing in acellula ROS concen a-
84
ions. On he o he hand, he inc eased pe oxide and supe oxide
85
concen a ions obse able unde H
2
O
2
and MSB exposu es could
86
no be sepa a ed om GSH/GSSG edox imbalances a any s esso
87
concen a ion es ed. The dis u bance o he GSH/GSSG edox bal-
88
ance unde H
2
O
2
and MSB- ea men s was explained by he ela-
89
i ely weak ca alase p oduc ion o A. nidulans, which bu dened
90
he GSH-dependen enzyma ic and non-enzyma ic ROS elimina-
91
ion pa hways (Pócsi e al., 2005).
92
The physiological e ec s o MSB a e no limi ed o he cyclic gen-
93
e a ion o O
2
because hese anions des oy 4Fe-4S p o eins, which
94
leads o he o ma iono dele e iousOH
adicals,and hede oxifica-
95
ion o MSB ca alyzed by glu a hione S- ans e ase also a ec s di-
96
ec ly he GSH pool o he cells (Toledano e al., 2003; Pócsi e al.,
97
2004). In addi ion, menadione can chemically modi y (a yla e) cell
98
componen s and enhance memb ane fluidi y (She ze e al.,
99
1992).MSBis he e o e likely oini ia emixedoxida i e/non-oxida-
100
i e s ess when employed a high (abo e 0.2 mmol l
1
) concen a-
101
ions and o sho pe iods o ime in ungal cul u es (Pócsi e al.,
102
2005).I is ema kable ha ashi omamixed- ypes ess esponse
103
owa ds a pu e oxida i e s ess esponse was obse ed unde long-
104
e m (6–9 h) exposu es o A. nidulans cul u es o MSB, when he
105
in acellula accumula ion o ROSand he dec ease in he GSH/GSSG
106
a io we e equally significan , and nume ous genes subjec ed o
107
supe oxide, pe oxide o GSH/GSSG-dependen ansc ip ional egu-
108
la ion we e esponding o oxida i e s ess (Pócsi e al., 2005).
109
A e comple ing he analysis o he da a ob ained om genome-
110
wide gene exp ession expe imen s, we add essed he ques ion o
111
whe he he la ge-scale and significan ansc ip ional changes
112
caused by MSB- ea men s would also esul in a p o eome signifi-
113
can ly di e en om ha o uns essed cul u es. Kim e al. (2008)
114
e iewed p o eomic da a collec ed in he Aspe gillii up o he yea o
115
2008 and epo ed only a combined o al o 28 cell su ace, 102
116
sec e ed and 139 in acellula p o eins ha ha e been iden ified in
117
10 di e en s udies. Taking in o conside a ion he p ac ical signifi-
118
canceo heseindus iallyandmedicallyimpo an ungiand he ac
119
ha mos o hem a e ully sequenced and hei genome anno a ions
120
ha e eached an ad anced le el (Wo man e al., 2009) hese num-
121
be s a e qui e modes . Because no p o eome s udy has been ca ied
122
ou ye in oxida i e s ess-exposed Aspe gillii, we would also ha e
123
liked o augmen ou p o eome-le el knowledge on he oxida i e
124
s ess de ense sys em o hese Euascomyce es (Miskei e al., 2009).
125
To compile da a o all he equi emen s, in his s udy we
126
mapped he in acellula soluble p o eome o A. nidulans ege a-
127
i e cells exposed o high-dose (0.8 mmol l
1
) MSB o long ime
128
pe iods (6 h). T ansla ional changes igge ed by oxida i e s ess
129
we e compa ed o genome-wide ansc ip ional changes eco ded
130
using EST-DNA-mic oa ays and flip-flop and dend ime cDNA
131
popula ion labeling echniques unde he same expe imen al con-
132
di ions (Pócsi e al., 2005).
133
2. Ma e ials and me hods
134
2.1. S ain, cul u e condi ions
135
Aspe gillus nidulans FGSC 26 (biA1, eA1) was used h oughou
136
his s udy and was a gi o S. Rosén (Uni e si y o Lund, Sweden).
137
Vege a i e mycelium was cul i a ed in minimal ni a e medium
138
and was exposed o 0.8 mmol l
1
MSB o 6 h as desc ibed be o e
139
by Pócsi e al. (2005). MSB- ea ed mycelia we e washed wi h
140
ice-cold phospha e-bu e ed saline (0.9% w/ NaCl in 0.1 mol l
1
141
phospha e bu e , pH 7.4) and dis illed wa e , and we e s o ed o-
142
zen a 20 °C in lysis bu e (20 mmol l
1
T is–HCl, pH 7.6,
143
10 mmol l
1
NaCl, 0.5 mmol l
1
deoxychola e) o p o eomics s ud-
144
ies. In DNA mic oa ay expe imen s, mycelial sample p epa a ion
145
and s o age we e pe o med as p e iously (Pócsi e al., 2005).
146
2.2. P o eomics s udies
147
In acellula soluble p o ein sample p epa a ion was ca ied ou
148
acco ding o Nandakuma and Ma en (2002) wi h some modifica-
149
ions. F ozen mycelia we e dis up ed wi h X-p ess (AB Biox,
150
Ge many), and he endogenous p o eases we e inac i a ed by
151
40
l
lml
1
P o easeInhibi o Cock ail(Sigma–Ald ich).Thecelldeb-
152
issuspensionwas cen i uged (6000 g, 4 °C,10 min),and he supe -
153
na an was ea ed s epwise by 7
l
lml
1
RNase/DNase/Mg mix
154
(0.25 mg ml
1
RNase, 0.5 mg ml
1
DNase, 50 mmol l
1
MgCl
2
;
155
0°C; 5 min) and an equal olume o 20% TCA (0 °C; 30 min). P ecip-
156
i a ed p o eins we e sepa a ed by cen i uga ion (6000 g, 4 °C,
157
20 min), and he pelle s we e washed wice wi h ice-cold ace one
158
and we e ai -d ied a oom empe a u e.
159
In wo-dimensional polyac ylamide gel elec opho esis (2D-
160
PAGE), p o ein samples (p o ein con en s we e se o 300
l
g, de e -
161
mined by he Non-In e e ing P o ein Assay Ki o Calbiochem) we e
162
applied on o 17 cm immobilized pH g adien (IPG) s ips (pH 5–8,
163
Bio-Rad) by passi e ehyd a ion o 12 h in a solu ion con aining
164
7 mol l
1
u ea, 2 mol l
1
hiou ea, 2% (w/ ) CHAPS, 50 mmol l
1
165
DTT and 0.5% ampholy e (Bio-Ly e 3/10 Ampholy e). Isoelec ic
166
ocusing(IEF)waspe o medinaP o eanIEFCell(Bio-Rad)applying
167
he ollowing ol agese ings:0–250 V o 20 min,250–10,000 V o
168
2.5 h and, in he final phase, 10,000 V o 8 h. The ea e , he IPG
169
s ips we e consecu i ely incuba ed in solu ions A and B o
170
20 mineach ime o educeandalkyla e hep o eins.Solu ionAcon-
171
ained 50 mmol l
1
T is/HCl, pH 8.8, 6 mol l
1
u ea, 30% ( / ) glyc-
172
e ol, 5% (w/ ) SDS and 2% (w/ ) DTT, when in solu ion B DTT was
173
eplaced by 6% (w/ ) iodoace amide. The second dimension o 2D-
174
PAGE was pe o med on 10–14% g adien SDS polyac ylamide gels
175
using he P o ean II xi Mul i-Cell (Bio-Rad). Gels we e s ained wi h
176
Ru henium II T is (Rabilloud e al., 2001; Lamanda e al., 2004) and
177
Coomassie B illian Blue.
178
Images o he 2D-PAGE gels we e gene a ed using a Ve saDoc
179
4000 imaging sys em (Bio-Rad), and he analysis o he 2D-images
180
was pe o med wi h he PDQues so wa e (Bio-Rad). P o ein sam-
181
ples coming om h ee independen expe imen s o each g ow h
182
condi ion we e analyzed in sepa a e 2D-PAGE uns, and he signi -
183
icances o he di e ences in he densi ome ic da a gained in MSB-
184
ea ed and con ol samples o indi idual p o eins we e es ima ed
185
by he S uden ’s - es .
186
P o ein spo s wi h significan ly highe op ical densi ies han
187
hei coun e pa s in ei he he s ess-exposed o he con ol cul-
188
u es we e cu om 2D-PAGE gels, diced, and hen we e insed wi h
189
25 mmol l
1
NH
4
HCO
3
{p epa ed in 50% ( / ) ace oni ile/wa e } o
190
emo e SDS and Coomassie B illian Blue. The p o eins in he spo s
191
we e diges ed wi h side-chain-p o ec ed po cine ypsin (P omega,
192
Madison, WI, USA; 25 mmol l
1
NH
4
HCO
3,
37 °C, 4 h), and he mass
193
spec ome ic analysis o he yp ic diges s was pe o med by on-
194
line LC/MSMS using a 3D ion ap (LCQ Flee , The mo Fishe Scien-
195
ific GmbH, B emen, Ge many) connec ed wi h a nanoHPLC sys em
196
(Mic oP o, Eldex, USA) and an au osample (Endu ance, Sunch om,
197
Ge many). Pep ide ac iona ions we e pe o med using a 3
l
m
198
A lan is™ dcC
18
column (75
l
m100 mm; Wa e s, Mil o d, MA,
199
USA), equilib a ed in 10% ( / ) aqueous solu ion o ace oni ile,
200
which con ained 0.1% o mic acid. A e sample injec ion, he
2T. Pusz ahelyi e al./Fungal Gene ics and Biology xxx (2010) xxx–xxx
YFGBI 2267 No. o Pages 12, Model 5G
30 Augus 2010
Please ci e his a icle in p ess as: Pusz ahelyi, T., e al. Compa ison o ansc ip ional and ansla ional changes caused by long- e m menadione exposu e
in Aspe gillus nidulans. Fungal Gene . Biol. (2010), doi:10.1016/j. gb.2010.08.006
201
concen a iono ace oni ile was inc eased o50%o e 40 min (0.1%
202
o mic acid, 300 nl min
1
flow a e). The mass spec ome e was
203
ope a ed in iple play mode: su ey scans we e ollowed by a 6-
204
Da-zoom scan and CID analysis on he mos abundan ion in he su -
205
ey. Singly cha ged ions we e excluded om he p ecu so selec-
206
ion; and dynamic exclusion was enabled. The MS/MS da a we e
207
p ocessed wi h Masco Dis ille ( e sion 2.1.1.0.) wi h peak picking
208
pa ame e s ecommended o ion ap da a.
209
The gene a ed peak-lis s we e submi ed o da abase sea ches
210
wi h Masco (in-house se e 2.2.04.) agains he Na ional Cen e
211
o Bio echnology In o ma ion (NCBI; www.ncbi.nlm.nih.go /)
212
non- edundan da abase ( elease 09-26-2007; 55,19,594 se-
213
quences). Sea ch pa ame e s we e se o 0.6 Da mass accu acy o
214
he p ecu so ion and 1.0 Da o he agmen ions. Only yp ic
215
clea ages we e conside ed and one missed clea age was pe mi ed.
216
Ca bamidome hyla ion o Cys- esidues was conside ed as fixed
217
modifica ion, while me hionine oxida ion, p o ein N-ace yla ion
218
andpy oglu amicacid o ma ion omN- e minalGln esidueswe e
219
ega ded as a iable modifica ions. The cu -o sco e, de e mined by
220
Masco using a 0.05significance h eshold(p60.05),was54.Tofind
221
he exac ORF ID codes and he unc ions o he p o eins, he se-
222
quences iden ified om he yp ic diges s we e analyzed wi h he
223
blas psea chp og amo Al schule al. (1997)in heAspe gillus Gen-
224
ome Da abase (www.b oad.mi .edu/). Whene e ‘‘hypo he ical p o-
225
eins” wi h no p edic ed unc ion we e iden ified, homology sea ch
226
was also ca ied ou ia ansla ed ORF que y e sus p o eins in NCBI
227
BLAST. Homology da a we e fil e ed acco ding o he 1E-40 expec a-
228
ion alue (E) cu o c i e ia.
229
Unless o he wise indica ed, p o eins wi h a leas ou iden i-
230
fied pep ides and wi h significan homologies equal o o abo e
231
he cu -o sco e 54, and/o wi h an a leas 20% p o ein sequence
232
co e age (Raman e al., 2005) a e p esen ed in his wo k. In he
233
high and low molecula mass anges, some p o eins wi h a leas
234
wo iden ified pep ides and lowe sequence co e age we e also ac-
235
cep ed. On he basis o Aspe gillus Genome Da abase (www.b oad.-
236
mi .edu/), heo e ical isoelec ic poin (pI) and molecula mass
237
(kDa) we e calcula ed o each p o ein wi h he Compu e pI/kDa
238
ool (Bjellq is e al., 1993, 1994; Gas eige e al., 2005;h p://
239
ca.expasy.o g/ ools/pi_ ool.h ml). Biochemical pa hway in o ma-
240
ion was ex ac ed om he Kyo o Encyclopedia o Genes and Gen-
241
omes (KEGG; e sion 51.0; elease July 1, 2009; h p://
242
www.genome.jp/;Kanehisa and Go o, 2000). The unc ional clus-
243
e ing o he p o eins was ca ied ou using he AmiGO Gene On ol-
244
ogy Da abase (h p://amigo.geneon ology.o g/cgi-bin/amigo/
245
go.cgi; elease Augus 27, 2009; Ca bon e al., 2009). FUN genes
246
we e analyzed o pu a i e domains in he Conse ed Domain
247
Da abase o he NCBI (Ma chle -Baue e al., 2005;h p://
248
www.ncbi.nlm.nih.go /si es/en ez?db=cdd).
249
2.3. Genomics s udie
250
Double p in ed EST-based DNA chips (3533 unique PCR-ampli-
251
fied p obes p in ed in 2 4073 spo s; Pócsi e al., 2005) we e used
252
o moni o changes in cDNA popula ions p epa ed om mRNA
253
pools isola ed om MSB-exposed and un ea ed con ol cul u es.
254
The ull desc ip ion o gene p obes including PCR p ime s, Okla-
255
homa S a e Uni e si y con ig IDs (OSU con ig IDs; PipeOnline
256
[h p://bioin o.oks a e.edu/pipeonline/]) and B oad Ins i u e (Cam-
257
b idge, MA, USA) ORF IDs (B oad Ins i u e Aspe gillus nidulans Da a-
258
base, h p://www.b oad.mi .edu/anno a ion/ ungi/aspe gillus/) a e
259
gi en a NCBI Gene Exp ession Omnibus (NCBI GEO; h p://
260
www.ncbi.nlm.nih.go /geo/) on Pla o ms GPL1752 and GPL1756.
261
Fluo escence labeling o he cDNA popula ions was ca ied ou
262
ollowing he ‘‘flip-flop” p o ocol o Hedge e al. (2000), whe e
263
Cy5-dUTP and Cy3-dUTP a e inco po a ed in o he cDNAs du ing
264
he e e se- ansc ip ion o he mRNA pools ex ac ed om
265
s ess-exposed and con ol cul u es, espec i ely (‘‘flip”), o ice
266
e sa (‘‘flop”). A e hyb idizing he cDNA pools on o he mic o-
267
a ays (Hegde e al., 2000; Pócsi e al., 2005), gene exp ession le els
268
cha ac e ized by fluo escence in ensi ies we e ead wi h a GenePix
269
4000B mic oa ay scanne (Axon Ins umen s), and he in ensi y
270
a ios we e calcula ed wi h GenePix P o 3.0 so wa e (Pócsi e al.,
271
2005).
272
De ec ed spo s wi h alse eadings we e fil e ed ou manually,
273
and da a poin s wi h backg ound mean +1 SD highe han he spo
274
in ensi y means o bo h dyes we e also dis ega ded (Pócsi e al.,
275
2005). Following ha , he backg ound-co ec ed a ios and log
2
a-
276
ios (M) o spo in ensi ies we e calcula ed, and he M alues we e
277
subjec ed o LOESS- ype block-by-block no maliza ion (Leung and
278
Ca alie i, 2003) using SAS o Windows, e sion 8 (SAS Ins i u e
279
Inc., Ca y, NC, USA) so wa e. In u he da a p ocessing, no mal-
280
ized log
2
a ios (M
0
) we e analyzed. Only gene p obes wi h M
0
al-
281
ues abo e o below he [+1; 1] M
0
h esholds alue (‘ wo old
282
ule’; Schena e al., 1996) we e conside ed o espond o MSB- ig-
283
ge ed oxida i e s ess. All DNA mic oa ay da a we e deposi ed in
284
NCBI GEO (h p://www.ncbi.nlm.nih.go /geo/index.cgi) on Pla -
285
o m GPL1752 in Folde GSE4713 (flip-flop da abase).
286
3. Resul s and discussion
287
3.1. mRNA and p o ein abundances in oxida i e s ess-exposed
288
A. nidulans
289
Compa ing p o ein concen a ions and mRNA exp ession le els
290
is a an ad anced le el in yeas esea ch bu lagging in filamen ous
291
ungi including he Aspe gillii (Kim e al., 2008). Yeas -based models
292
a e o p ima y impo ance when he easons o he poo co ela-
293
ions be ween mRNA and p o ein le els ypically ound in euka y-
294
o ic cells a e s udied and discussed (Gygi e al., 1999; de Nobel
295
e al., 2001; G eenbaum e al., 2003; Beye e al., 2004; B ockmann
296
e al., 2007; Schmid e al., 2007; Tulle e al., 2007; de G oo e al.,
297
2007). The co ela ion depends on bo h he cellula localiza ion
298
and he physiological unc ion o he p o eins (G eenbaum e al.,
299
2003; Beye e al., 2004; Schmid e al., 2007; de Godoy e al.,
300
2008;Rossignole al.,2009),andisinfluencedbymanycomplex ac-
301
o s including ansla ional ac i i y (B ockmann e al., 2007), p o-
302
ein hal -li es (Beye e al., 2004; Belle e al., 2006) as well as
303
na u al and manu ac u ed sys ema ic noise (G eenbaum e al.,
304
2003). I is impo an o no e ha da a gained by ORF (EST) based
305
DNA mic oa ays may be dis o ed o some ex en by c oss-hyb id-
306
iza ions(Iwahashie al.,2007),whichmay also influence he con o -
307
mi y o he p o eome and ansc ip ome da a.
308
Simila o gene al and specific s ess esponses, which a e well-
309
desc ibed a he le el o ansc ip ion, pos - ansc ip ional gene al
310
and specific s ess esponses also exis in yeas (B ockmann e al.,
311
2007). Many s ess- esponsi e genes a e subjec ed o he pos - an-
312
sc ip ional egula ion mechanism ‘‘ ansla ion on demand” (Beye
313
e al., 2004; B ockmann e al., 2007), which is c ucially impo an
314
when adap ing o an en i onmen al s ess ha equi es a quick cel-
315
lula esponse (B ockmann e al., 2007). As a consequence, changes
316
in he exp ession o mRNA popula ions do no necessa ily co ela e
317
wi h he le els o he ansla ed p o eins and ice e sa (Beye e al.,
318
2004; Kolkman e al., 2006; Tulle e al., 2007). In gene al, ansc ip-
319
ion ac o sandsignalinggenesa e egula edmainlypos - ansc ip-
320
ionally (B ockmann e al., 2007) while many elemen s o he
321
biosyn he ic pa hways a e con olled ansc ip ionally (B o e al.,
322
2003; Washbu n e al., 2003; Rossignol e al., 2009).
323
Because he applicabili y o yeas -based models in he desc ip-
324
ion o Aspe gillus s ess esponse sys ems was limi ed (Miskei
325
e al., 2009) ou p ima y goal was o gain in o ma ion on he
326
co ela ion be ween p o ein and mRNA abundances in oxida i e
327
s ess-exposed A. nidulans cells.
T. Pusz ahelyi e al./ Fungal Gene ics and Biology xxx (2010) xxx–xxx 3
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in Aspe gillus nidulans. Fungal Gene . Biol. (2010), doi:10.1016/j. gb.2010.08.006
328
P o eome analysis o MSB-exposed (6 h) A. nidulans cul u es e-
329
ealed 82 s ess- ela ed in acellula p o eins unde going signifi-
330
can changes (Fig. 1;Supplemen a y 1). Ou o hose, 17 p o eins
331
we e de ec ed in mo e han one spo and fi e o hem we e iden-
332
ified in ep essed and induced o ms as well (Supplemen a y 1;
333
Table 1). A su ey o he li e a u e and he Aspe gillus S ess Da a-
334
base (h p://193.6.155.82/Aspe gillusS ess/;Miskei e al., 2009)
335
e ealed ha me ely 19 o he p o eins had been ela ed o any
336
kind o s ess esponse hus a (e.g. oxida i e, hea , osmo ic s ess,
337
un olded p o ein esponse; Supplemen a y 2). DNA mic oa ay
338
da abases gained wi h flip-flop ( his s udy) and dend ime (Pócsi
339
e al., 2005) cDNA labeling echniques p o ided us wi h ansc ip-
340
ion da a o he genes encoding 42 o he 82 iden ified s ess- e-
341
sponse p o eins (Supplemen a y 3). We ound bo h flip-flop and
342
dend ime mic oa ay da a o he g ea majo i y o hese genes
343
(38 o he 42), and he DNA chips used in hese s udies con ained
344
mo e han one di e en PCR-amplified p obes o 17 s ess- ela ed
345
genes (Supplemen a y 3).
346
When he co ela ion be ween ansc ip ome and p o eome
347
da ase s was examined, coincidence be ween p o ein le els and
348
gene exp essions was ound wi h 6 h MSB- ea ed p o eome (P)
349
and 6 h ea ed ansc ip ome samples. Coincidence le els wi h
350
flip-flop-labeled (F) and dend ime -labeled (D6) ansc ip ome
351
da ase s we e 40% and 34% (Fig. 2, Panels P–F and P-D6, II + RR),
352
espec i ely. These coincidence coe ficien s we e in good acco -
353
dance wi h p e ious obse a ions co ela ing mRNAs wi h p o ein
354
abundance (Tian e al., 2004; Nie e al., 2006; B ockmann e al.,
355
2007). Poo coincidence coe ficien s (14–17%) we e ound compa -
356
ing 6 h MSB- ea ed p o eome and 0.5–3 h dend ime da a (Fig. 2,
357
Panels P-D0.5, P-D1 and P-D3, II + RR). This may be a consequence
358
o he ela i ely slowly accumula ing oxida i e s ess in MSB-ex-
359
posed A. nidulans cells (Pócsi e al., 2005). The con o mi y be ween
360
p o eome and ansc ip ome da a was 29% when p o eome was
361
compa ed o pooled dend ime da a (Fig. 2, Panel P-D0.5–6,
362
II + RR), and he lowes pe cen age (3%) o opposi e p o eome
363
and ansc ip ome changes was eco ded wi h 3 and 6 h ansc ip -
364
omes (Fig. 2, Panels P-D3 and P-D6, IR + RI). The pe cen age o p o-
365
eome changes no eflec ed in he a ia ions o he ansc ip ome
366
was highe in he dend ime -based DNA mic oa ay hyb idiza ions
367
(54–79%; Fig. 2, Panels P-D0.5, P-D1, P-D3 and P-D6, IO + RO) han
368
in he flip-flop-based DNA mic oa ay hyb idiza ion (33%, Fig. 2,
369
Panel P–F, IO + RO).
370
An al e na ing p o ein exp ession pa e n was obse ed o he
371
glycoly ic pa hway enzymes AcuG ( ep essed), FbaA (induced),
372
GpdA ( ep essed), PgkA (induced), EnoA ( ep essed), PkiA (in-
373
duced) a e 6 h MSB- ea men s (Fig. 3). As shown be o e by Pócsi
374
e al. (2005), he exp essions o he glycoly ic pa hway genes acuG,
375
baA,gpdA and pkiA we e fluc ua ing (pe iodically ep essed and
376
induced) as a unc ion o he MSB-exposu e ime (Supplemen a y
377
3,Fig. 4A). Theo e ically, an al e na ing p o ein exp ession pa e n
378
may a ise in a me abolic pa hway when ansc ip ional and ans-
379
la ional changes a e synch onous o he indi idual genes and gene
380
p oduc s bu he equencies o hese fluc ua ions a e ma kedly
381
di e en (Pócsi e al., 2005). A simila phenomenon has al eady
382
been obse ed unde li hium ea men s o budding yeas cells,
383
when e e y second gene, namely PGM2 (pgmB o holog), FBP1
384
(acuG o holog), TDH1 (gpdA o holog) and GPM2, PYK2 (pkiA o ho-
385
log), was up- egula ed in he glycoly ic pa hway (B o e al., 2003).
386
Anae obiosis also a ec ed gene exp essions and p o ein p oduc-
387
ions in qui e di e en ways in he glycoly ic pa hway o S. ce e i-
388
siae because mos o he gene exp essions emained unchanged
389
bu he quan i ies o a significan numbe o gene p oduc s in-
390
c eased conside ably (de G oo e al., 2007).
391
Opinions on he egula ion o glycoly ic p o eins a e dissen ing.
392
These p o eins may be unde ansc ip ional egula ion because
393
genes in he unc ional ca ego ies ‘‘me abolism,” ‘‘ene gy,” and
394
‘‘p o ein syn hesis” exhibi he s onges co ela ion be ween
395
mRNA and p o ein le els in yeas s (Beye e al., 2004), and a mod-
396
e a e co ela ion be ween glycoly ic pa hway mRNA and p o ein
397
le els has been eco ded by Schmid e al. (2007). On he o he
398
hand, pos - ansc ip ional modula ions may also play an impo an
399
ole in he egula ion o he glycoly ic pa hway enzymes (de G oo
400
e al., 2007), and he specific ac i i ies o he me abolic enzymes
401
may also influence he obse ed p o ein le els (Schmid e al.,
402
2007). Based on ou s udy, fluc ua ing mRNA and al e na ing p o-
403
ein exp ession le els sugges a ema kably flexible egula ion o
404
he glycoly ic pa hway enzymes in s ess-exposed A. nidulans.
405
I is wo h no ing ha gene exp ession fluc ua ions a e no lim-
406
i ed o glycoly ic pa hway genes as demons a ed by DNA mic o-
407
a ay expe imen s (Table 1;Supplemen a y 3;Pócsi e al., 2005),
408
No he n blo hyb idiza ions (Pócsi e al., 2005) and eal- ime e-
409
e se- ansc ip ion polyme ase chain eac ion assays (Supplemen-
410
a y 4), and such fluc ua ing gene exp ession pa e ns may also
411
explain, a leas in pa , he obse ed asynch ony o he ansc ip-
412
ome and p o eome da a (Fig. 2).
413
Ne e heless, o some genes and hei p o ein p oduc s an-
414
sc ip ional and ansla ional changes we e in good acco dance
415
(Supplemen a y 3), and he gene exp essions we e ei he consis-
416
en ly induced (e.g. genes encoding a pu a i e glu a hione S- ans-
417
e ase and a FUN p o ein o holog o A. umiga us AFUA_2G09530)
418
o ep essed (e.g. hsp70 and ungA;Table 1;Fig. 4B). S ess- e-
419
sponse genes wi h minimal a ia ions in hei mRNA exp ession
420
le els a e induc ion like Gs (Fig. 4B) we e egula ed mainly a
421
ansc ip ional le el and he p o ein concen a ions ended o be
422
less ‘‘noisy” in yeas (B ockmann e al., 2007). As a consequence,
423
mRNA le els co ela ed well wi h p o ein concen a ions in hese
424
cases (G eenbaum e al., 2003; Schmid e al., 2007).
425
3.2. S ess- esponsi e p o eins in MSB- ea ed A. nidulans
426
Analyzing he p o eins desc ibed wi h he GO e m ‘ esponse o
427
s ess’, he induced T xR hio edoxin educ ase, a fla ohemop o-
428
ein (ANID_07169.1) and a ni o educ ase (ANID_02343.1) e-
429
placed he ep essed pu a i e o holog o budding yeas ’s
430
mi ochond ial Ccp1 cy och ome C pe oxidase and he ep essed
431
pe oxi edoxins in he cen e o he oxida i e s ess de ense sys em
432
o MSB-exposed A. nidulans (Table 1;Fig. 3;Supplemen a y 2). The
433
appea ance o a fla ohemop o ein among he induced p o eins
434
may be indica i e o de eloping ni osa i e s ess in MSB- ea ed
435
A. nidulans mycelia simila o budding yeas cells (Table 1;Liu
436
e al., 2000; Te Biesebeke e al., 2009). In S. ce e isiae, bo h MSB
437
and H
2
O
2
ea men s ha e been shown o gene a e ni osa i e
438
s ess (Almeida e al., 2007; Oso io e al., 2007).
439
GSH is he cen e piece o he an ioxida i e de ense sys em in al-
440
mos all euka yo ic cells, including ungi. GSH is p esen in high
441
concen a ions in li ing cells, and is he majo ese oi o educed
442
non-p o ein sul u (Pócsi e al., 2004). In MSB-exposed A. nidulans
443
mycelium, he GSH concen a ion d ops and a numbe o GSH-bio-
444
syn he ic and GSH- egene a ing enzymes a e induced o main ain
445
a physiologically ele an GSH/GSSG balance (Pócsi e al., 2005).
446
The induc ion o isofla one educ ase is an indica o o he limi ed
447
a ailabili y o GSH in maize (Pe ucco e al., 1996) and, no su p is-
448
ingly, i s o holog (ANID_08815.1) was also induced in MSB- ea-
449
ed A. nidulans (Table 1), when he GSH/GSSG a io is
450
significan ly dec eased (Pócsi e al., 2005). Induced glu a hione-S-
451
ans e ases (Gs 3 and a pu a i e Gs ) we e also connec ed o he
452
oxida i e s ess esponse (Table 1) because hese enzymes a e e-
453
qui ed o p o ec euka yo ic cells om pe oxide-induced cell dea h
454
(Pócsi e al., 2005) and he dele e ious e ec s o menadione i sel
455
(Em i e al., 1999).
456
Unde long- e m, ch onic oxida i e s ess, glucose and ammo-
457
nia up ake a e educed in ungi (Em i e al., 1997; Oso io e al.,
4T. Pusz ahelyi e al./ Fungal Gene ics and Biology xxx (2010) xxx–xxx
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in Aspe gillus nidulans. Fungal Gene . Biol. (2010), doi:10.1016/j. gb.2010.08.006
458
2004; Li e al., 2008a) and, he e o e, cells ha e o cope wi h glu-
459
cose and ni ogen sho ages as well, in addi ion o he neu aliza-
460
ion o ROS and he main enance o he GSH/GSSG and NADP
+
/
461
NADPH edox balances (Zadzinski e al., 1998; Pócsi e al., 2005;
462
Li e al., 2008b). The app op ia e s ess- esponsi e egula ion o
463
he ca bon and ni ogen me abolic pa hways is o ca dinal impo -
464
ance in he oxida i e s ess de ense o s ess-exposed cells.
465
In good ag eemen wi h his, a ious enzymes o ca bon me ab-
466
olism cha ac e ized wi h he main GO e ms ‘‘hexose me abolism”
467
(inco po a ing glycolysis, gluconeogenesis, pen ose phospha e
468
shun ), ‘‘TCA cycle”, ‘‘alcohol me abolism” as well as ‘‘ca boxylic
469
acid me abolism”, ‘‘manni ol me abolism” and ‘‘amino acid me ab-
470
olism” we e ound o be s ess- esponsi e (Table 1). In he glyco-
471
ly ic pa hway, he main ATP-p oduce PkiA py u a e kinase and
472
PgkA 3-phosphoglyce a e kinase we e induced oge he wi h FbaA
473
uc ose 1,6-bisphospha e aldolase (Table 1,Fig. 3). I is impo an
474
o no e ha Pgk1p 3-phosphoglyce a e kinase and Fba1p uc ose
475
1,6-bisphospha e aldolase we e also up- egula ed in menadione-
6704
6201
5301
6104
5102
7303
2304
8404
5502 7507 8605
6706
4003
4501
3611
8408
7104
2102
4403
5403
2406
4705
8702
6502
4001
8303
5504
7706
3305
2602
4803
4503
8701
1108
2104
2505
5711
6402
7003
5508
9201
8206
7508
6005 7004
8411
3206
1103
3401
pH5 pH8
B
4608
6703
7305
4406
6711
7304
4704
3711
6724
8325
3610
7510
2506
3618
1803
6806
3513
6515
5507
5401
6409
2703
4807
6208
4505
4802
3509
8208
1310
2612
5807
8309
1114
3403
7111
7205
2107
5202
6603
3209 5216
4509
pH5 pH8
A
Fig. 1. 2D-PAGE sepa a ion and iden ifica ion o in acellula soluble s ess- esponsi e p o eins in MSB-exposed A. nidulans ege a i e cul u es. Pa s A and B ep esen
uns essed con ol and MSB- ea ed A. nidulans FGSC 26 cul u es, espec i ely. Spo s wi h significan ly induced (Pa B) o ep essed (Pa A) p o eins a e localized wi h
a ows and ma ked wi h spo ID (also lis ed in Supplemen a y 1). Only one o h ee independen uns is shown.
T. Pusz ahelyi e al./ Fungal Gene ics and Biology xxx (2010) xxx–xxx 5
YFGBI 2267 No. o Pages 12, Model 5G
30 Augus 2010
Please ci e his a icle in p ess as: Pusz ahelyi, T., e al. Compa ison o ansc ip ional and ansla ional changes caused by long- e m menadione exposu e
in Aspe gillus nidulans. Fungal Gene . Biol. (2010), doi:10.1016/j. gb.2010.08.006
Table 1
Oxida i e s ess- esponsi e p o eins in MSB-exposed Aspe gillus nidulans.
Func ions
a
A. nidulans
locus ID
b
P o eomics
c
Genomics
c
Flip-
flop
6h
Dend ime
0.5 h
Dend ime
1h
Dend ime
3h
Dend ime
6h
Dend ime
0.5–6 h
Response o s ess
1-Cys pe oxi edoxin, pu a i e ANID_10223.1 R
Pe oxi edoxin P xA ANID_08692.1 R
cy och ome c pe oxidase Ccp1 ANID_10220.1 R
Fla ohemop o ein ANID_07169.1 I I R R A A
Ni o educ ase ANID_02343.1 I
Thio edoxin educ ase T xR ANID_03581.1 I
Glu a hione S- ans e ase Gs B ANID_06024.1 I I I 0 0 I I
Glu a hione S- ans e ase Gs 3 ANID_10273.1 I 0
Hexose me abolism
UDP-glucose-4-epime ase GalGb ANID_04727.1 R
Phosphoglucomu ase PgmB ANID_02867.1 R R 0 0
F uc ose-1,6-bisphospha ase AcuG ANID_05604.1 R I I 0 I 0 I
F uc ose 1,6-bisphospha e aldolase FbaA ANID_02875.1 I 0 0 0 0 0 0
Glyce aldehyde-3-phospha e dehyd ogenase GpdA ANID_08041.1 R I 0 0 0 0 0
3-Phosphoglyce a e kinase PgkA ANID_01246.1 I
Enolase EnoA (AcuN) ANID_05746.1 R
Py u a e kinase PkiA ANID_05210.1 I I 0 R I I A
Glucose-6-phospha e 1-dehyd ogenase GsdA ANID_02981.1 I I 0 I 0 I
Ribose 5-phospha e isome ase ANID_05907.1 I I 0 R 0 I A
T anske olase ANID_09180.1 R
T ansaldolase PppA ANID_00240.1 I R 0 R 0 R R
T ica boxylic acid cycle
Aconi ase AcoA ANID_05525.1 R 0 0 0 0 0 0
Hypo he ical p o ein simila o isoci a e dehyd ogenase
subuni 2 IdpA
ANID_01003.1 I 0 0 0 0 0 0
Mi ochond ial mala e dehyd ogenase MdhA ANID_06717.1 R
Mala e dehyd ogenase, MdhC ANID_06499.1 R 0 0 0 0 0 0
Alcohol me abolism
Aldehyde dehyd ogenase AldA ANID_00554.1 R 0 I R 0 0 A
Zinc-con aining alcohol dehyd ogenase ANID_02351.1 I
Alcohol dehyd ogenase ANID_08406.1 I
Ca boxylic acid me abolism
Py u a e deca boxylase PdcA ANID_04888.1 R A 0 R 0 R R
NAD-dependen o ma e dehyd ogenase AciA ANID_06525.1 R 0 0 0 R R
Manni ol me abolism
Manni ol 2-dehyd ogenase ANID_07590.1 I I R 0 0 0 R
Cellula amino acid me abolism
A gininosuccina e syn he ase ANID_01883.1 R
Fuma ylace oace a e hyd olase FahA ANID_01896.1 R
Alanine ansaminase ANID_01923.1 A
L
-o ni hine amino ans e ase O aA ANID_01810.1 I R
O ni hine ca bamoyl ans e ase A gB ANID_04409.1 R I 0 0 0 0
Dihyd oxy-acid dehyd a ase ANID_06346.1 I
Cys a hionine be a-syn hase MecA ANID_05820.1 I
3-Phosphose ine amino ans e ase ANID_10298.1 R
NADP-specific glu ama e dehyd ogenase GdhA ANID_04376.1 A
Glu amine syn he ase GlnA ANID_04159.1 R I R 0 0 R
Choline oxidase (CodA), pu a i e ANID_01429.1 I
Phospha idyl syn hase [Aspe gillus umiga us A 293] NCBI ANID_05564.1 I I 0 I I 0 I
Glucose–me hanol–choline oxido educ ase ANID_08547.1 R
Cellula lipid me abolism
Myo-inosi ol-1-phospha e syn hase ANID_07625.1 I I R 0 0 0 A
Ace yl-CoA ace yl ans e ase, pu a i e ANID_01409.1 R
Ribofla in biosyn hesis
6,7-Dime hyl-8- ibi yl-lumazine syn hase RiboG ANID_10718.1 R R R 0 0 R R
GTP cyclohyd olase II ANID_10981.1 R R
Cy oskele on o ganiza ion
Hypo he ical p o ein simila o fimb in FimA ANID_05803.1 I 0 0 0 0 0 0
Chi in biosyn hesis
UDP-N-ace ylglucosamine py ophospho ylase UngA ANID_09094.1 R 0 R 0 0 R R
Nucleo ide sal age
Adenine phospho ibosyl ans e ase 1 ANID_09083.1 R
Gene a ion o p ecu so me aboli es and ene gy
Ubiquinol–cy och ome c educ ase i on–sul u subuni ANID_02306.1 I
Ino ganic py ophospha ase IppA ANID_02968.1 R
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YFGBI 2267 No. o Pages 12, Model 5G
30 Augus 2010
Please ci e his a icle in p ess as: Pusz ahelyi, T., e al. Compa ison o ansc ip ional and ansla ional changes caused by long- e m menadione exposu e
in Aspe gillus nidulans. Fungal Gene . Biol. (2010), doi:10.1016/j. gb.2010.08.006
476
exposed S. ce e isiae cells (Kim e al., 2007a). As demons a ed by
477
Pócsi e al. (2005), he exp ession o some genes encoding glyco-
478
ly ic enzymes was esponsi e o GSH/GSSG edox imbalance, e.g.
479
FbaA was ep essed conside ably, and his migh esul in he
480
in acellula accumula ion o uc ose-1,6-bisphospha e, a mi o-
481
chond ion-p o ec an me aboli e (Pócsi e al., 2005). The p o eome
482
da a challenged his hypo hesis because FbaA was clea ly induced
483
in MSB-exposed cul u es (Table 1).
484
In he Aspe gillus S ess Da abase (Miskei e al., 2009), GsdA
485
glucose-6-phospha e 1-dehyd ogenase, AcuG uc ose-1,6-bis-
486
phospha ase and GalGb UDP-glucose-4-epime ase om hexose
487
me abolic enzymes a e indica ed as s ess- ela ed p o eins (Sup-
488
plemen a y 2). Mo eo e , some da a published ea lie on GpdA
489
glyce aldehyde-3-phospha e dehyd ogenase, EnoA enolase and
490
hei yeas o hologs unde lined he impo ance o hese enzymes
491
in e sa ile s ess esponses. Fo example, ungal glyce aldehyde-
492
3-phospha e dehyd ogenases we e epo ed o pa icipa e in
493
osmoadap a ion (Kim e al., 2007b), in ci ic acid s ess (Law ence
494
e al., 2004), as well as in he esponse o concanamycin (Melin
495
e al., 2002) o ampho e icin B (Yu e al., 2007) ea men s, and
496
enolases a e also well-known pa icipan s in a ious s ess e-
497
sponses (Hu e al., 2003; Re e e -B ancha e al., 2004; En elis
498
e al., 2006; Kwon e al., 2009; Pandey e al., 2009). GAPDH, he
499
budding yeas o holog o GpdA, was a a ge o ex ensi e p o eol-
500
ysis unde ex ended (200 min) H
2
O
2
ea men , unde wen S-ni -
501
osyla ion and en e ed o he nucleus whe e i induced apop osis
502
(Almeida e al., 2007).
503
A sa is ac o y NADPH p oduc ion is o pi o al impo ance in he
504
main enance o he GSH, glu a edoxin and hio edoxin-dependen
505
elemen s o he an ioxidan de ense sys em (Juhnke e al., 1996). In
506
compliance wi h he NADPH equi emen o he s ess-exposed
507
cells, he main NADPH-p oduce enzymes GsdA and isoci a e
508
dehyd ogenase we e induced.
509
As a as he ni ogen me abolism is conside ed, wo key en-
510
zymes o ‘‘cellula amino acid me abolism” we e also iden ified;
511
GdhA NADP-specific glu ama e dehyd ogenase was ound in h ee
Table 1 (con inued)
Func ions
a
A. nidulans
locus ID
b
P o eomics
c
Genomics
c
Flip-
flop
6h
Dend ime
0.5 h
Dend ime
1h
Dend ime
3h
Dend ime
6h
Dend ime
0.5–6 h
Signal ansduc ion
G-p o ein complex be a subuni CpcB ANID_04163.1 A 0 R I 0 0 A
T ansla ion
Hypo he ical p o ein simila o elonga ion ac o EF-Tu ANID_01084.1 I
Elonga ion ac o 2 ANID_06330.1 R
T ansla ion elonga ion ac o eEF-1B gamma subuni El A ANID_09304.1 I
His idyl– RNA syn he ase ANID_00046.1 I I 0 0 R R
Aspa yl– RNA syn he ase Dps1 ANID_04550.1 R R 0 I R A
P o oplas sec e ed p o ein 2 [Aspe gillus e eus NIH2624]NCBI ANID_00297.1 I
RNA binding p o ein [Aspe gillus umiga us A 293] NCBI ANID_05480.1 I I I 0 I I I
P o ein olding, in acellula anspo
Pep idyl–p olyl cis– ans isome ase D Cp 6 ANID_04583.1 R 0 0 0
Hsp70 ANID_05129.1 R R R 0 0 R R
GTP-binding nuclea p o ein ANID_05482.1 R I 0 0 0 0 0
P o ein ca abolism
Hypo he ical p o ein simila o p o easome egula o y subuni 8 ANID_05121.1 I
P o easome componen P e6 ANID_08054.1 I
Unknown biological p ocess
Oxido educ ase ANID_00179.1 A
Oxido educ ase, hypo he ical ANID_00895.1 A R 0 0 I R A
Zinc-binding oxido educ ase ANID_10098.1 I
NADH:fla in oxido educ ase/NADH oxidase ANID_05228.1 I
NADH-dependen fla in oxido educ ase ANID_06753.1 I
Zinc-binding oxido educ ase ToxD ANID_11094.1 I
NAD binding Rossmann old oxido educ ase ANID_02208.1 I
Isofla one educ ase amily p o ein [Aspe gillus umiga us
A 293] NCBI
ANID_08815.1 I I 0 0 0 I I
Be a-lac amase amily p o ein ANID_05422.1 R R 0 0 0 0 0
Conse ed hypo he ical p o ein wi h homology o
me hyl ans e ase [Ajellomyces de ma i idis ER-3] NCBI
ANID_02561.1 I
NAD dependen epime ase/dehyd a ase ANID_05989.1 R
FUN p o eins
FUN; e a icopep ide epea domain-con aining p o ein ANID_03987.1 I
FUN; UPF0160 domain-con aining p o ein MYG1 ANID_04178.1 R
FUN; DUF833 domain-con aining p o ein ANID_06058.1 I 0 0 I I I I
FUN; DUF636 domain-con aining p o ein ANID_07594.1 I 0 0 0
FUN ANID_10219.1 I 0 0 0 0 0 0
FUN ANID_10260.1 I 0 R 0 0 R
a
Pu a i e o e ified physiological unc ions o he s ess- esponse p o eins iden ified in he p o eomics s udies. Physiological unc ions we e ex ac ed om he Aspe gillus
Compa a i e Da abase (h p://www.b oadins i u e.o g/anno a ion/genome/aspe gillus_g oup/Mul iHome.h ml), he Cen al Aspe gillus Da a REposi o y CADRE (Mabey e al.,
2004;h p://www.cad e-genomes.o g.uk/), he Aspe gillus Genome Da abase (h p://www.aspe gillusgenome.o g/), he Gene On ology Da abase (h p://amigo.geneon olo-
gy.o g/cgi-bin/amigo/go.cgi) and he Saccha omyces Genome Da abase (SGD, h p://www.yeas genome.o g/).
b
A. nidulans locus ID om he Aspe gillus Compa a i e Da abase (h p://www.b oadins i u e.o g/anno a ion/genome/aspe gillus_g oup/Mul iHome.h ml).
c
Le e s I, R, 0 and A s and o ‘‘significan ly induced”, ‘‘significan ly ep essed”, ‘‘no significan induc ion o ep ession” and ‘‘ambi alen change”, espec i ely. Fo u he
explana ion o he A ‘‘ambi alen change” ca ego y in ei he he genomics o he p o eomics s udies, see he cap ion o Fig. 2. A summa y o he changes in he gene
exp ession le els can be ead in Supplemen a y 3.
T. Pusz ahelyi e al./ Fungal Gene ics and Biology xxx (2010) xxx–xxx 7
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Please ci e his a icle in p ess as: Pusz ahelyi, T., e al. Compa ison o ansc ip ional and ansla ional changes caused by long- e m menadione exposu e
in Aspe gillus nidulans. Fungal Gene . Biol. (2010), doi:10.1016/j. gb.2010.08.006
512
spo s, and he enzyme was induced in wo o hem unde MSB-
513
s ess meanwhile GlnA glu amine syn he ase was ep essed (Table
514
1;Fig. 3;Supplemen a y 2). The pos - ansc ip ional egula ion o
515
budding yeas ’s GDH1 (o holog o GdhA) was obse ed by se e al
516
au ho s (Dang e al., 1996; DeLuna e al., 2001; G i fin e al., 2002;
517
Riego e al., 2002; Kolkman e al., 2006), and he appea ance o
518
mul iple GdhA spo s (bo h induced and ep essed) on he 2D-PAGE
519
gels is in good ag eemen wi h hese obse a ions. Impo an ly, he
520
ansc ip ion o GdhA was ep essed by glucose, induced by ni o-
521
gen limi a ion (Kolkman e al., 2006) and up- egula ed unde hyp-
Fig. 2. Compa ison o p o ein and mRNA le els in MSB-exposed A. nidulans. The compa ison was accomplished on s ess- esponse p o eins (P; Table 1) and gene exp ession
da abases ob ained wi h dend ime (D) and flip-flop (F) labeling DNA mic oa ay echnology (Supplemen a y 3;Pócsi e al., 2005). D0.5, D1, D3 and D6 s and o mic oa ay
da a eco ded unde 0.5, 1, 3 and 6 h exposu es o MSB (Supplemen a y 3;Pócsi e al., 2005). D0.5–6 symbolizes a unified da ase o he 0.5–6 h dend ime DNA mic oa ay
expe imen s. Ma ks I, R, 0 and A s and o ‘‘significan ly induced”, ‘‘significan ly ep essed”, ‘‘no significan induc ion o ep ession” and ‘‘ambi alen change”, espec i ely.
The ‘‘ambi alen change” g oup included genes wi h ambiguous o e en opposi e ansc ip ional changes eco ded on di e en PCR-amplified gene p obes a he same MSB-
exposu e ime o wi h opposi e ansc ip ional changes eco ded on he same gene p obe a di e en MSB-exposu e imes. In p o eomic expe imen s, he ‘‘ambi alen
change” g oup included s ess- ela ed p o eins wi h opposi e changes in hei quan i ies eco ded in sepa a e p o ein spo s.
8T. Pusz ahelyi e al./ Fungal Gene ics and Biology xxx (2010) xxx–xxx
YFGBI 2267 No. o Pages 12, Model 5G
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Please ci e his a icle in p ess as: Pusz ahelyi, T., e al. Compa ison o ansc ip ional and ansla ional changes caused by long- e m menadione exposu e
in Aspe gillus nidulans. Fungal Gene . Biol. (2010), doi:10.1016/j. gb.2010.08.006
522
oxic condi ions (Shimizu e al., 2009). Changes in he S. ce e isiae
523
GLN1 glu amine syn he ase (o holog o GlnA) ansc ip and p o-
524
ein le els showed poo co ela ions in la ge-scale s udies (G i fin
525
e al., 2002; Washbu n e al., 2003), and opposi e ansc ip ional
526
changes we e also obse ed by us o GlnA in flip-flop (induc ion)
527
and dend ime ( ep ession) DNA mic oa ay expe imen s while
528
he p o ein le el was significan ly dec eased (Table 1).
529
The sul u con aining amino acid biosyn he ic pa hways we e
530
ep esen ed solely by MecA cys a hionine b-syn hase among he
531
s ess-induced p o eins (Table 1;Supplemen a y 2). MecA ca a-
532
lyzes he homocys eine/cys a hionine con e sion and, hence, plays
533
an impo an ole in he biosyn hesis o cys eine, one o he h ee
534
amino acids building up GSH (Pócsi e al., 2004). Cys eine can also
535
be syn hesized in an al e na i e pa hway, which includes cys eine
536
syn hase (cysB, ANID_08057.1) and cysB was up- egula ed unde
537
he MSB- ea men s (Pócsi e al., 2005). The e o e, bo h cys eine
538
biosyn he ic pa hways may ope a e in oxida i e s ess-exposed
539
A. nidulans hyphae. I is impo an o no e ha he cys a hionine
540
pa hway as well as GSH p oduc ion we e highly induced unde
541
cadmium s ess in yeas (Vido e al., 2001; Mendoza-Cóza l e al.,
542
2005; Baudouin-Co nu and Laba e, 2006) and in Blas ocladiella
543
eme sonii (Geo g and Gomes, 2007). In he la e species, only he
544
cys a hionine pa hway ope a es.
545
Two enzymes in he u ea cycle, A gB o ni hine ca bamoyl ans-
546
e ase and a ginosuccina e syn he ase, we e ep essed in he
547
o ni hine–ci ulline-
L
-a ginosuccina e biocon e sion pa hway,
548
howe e , O aA
L
-o ni hine amino ans e ase was induced, and his
549
may esul in he accumula ion o o ni hine and a subsequen in-
550
c ease in he glu ama e biosyn hesis. Because he TCA cycle was
551
ep essed a mala e dehyd ogenase and AcoA aconi ase (Table 1;
552
Fig. 3), he glu ama e equi emen o he GSH biosyn hesis may
553
be me by he O aA pa hway.
554
Ace yl-CoA C-ace yl ans e ase (ANID_01409.1), which is classi-
555
fied unde he GO e m ‘‘ a y acid me abolism” bu can be linked
556
o a ious me abolic pa hways including he syn hesis and deg a-
557
da ion o ke on bodies, aline, leucine, isoleucine, he deg ada ion
558
o lysine, he me abolisms o py u a e and yp ophan, was e-
559
p essed. Myo-inosi ol-1-phospha e syn hase in he biosyn hesis
560
o inosi ol phospholipids (Reynolds, 2009) and CodA, a pu a i e
561
choline oxidase in he biosyn hesis o he osmop o ec an glycine
562
be aine (Pa k and Gande , 1998; Bu g and Fe ais, 2008) we e in-
563
duced oge he wi h a phospha idyl syn hase (ANID_05564.1).
564
Unexpec ed da a we e ob ained on biosyn heses o i amins be-
565
cause wo enzymes, RiboG 6,7-dime hyl-8- ibi yl-lumazine syn-
566
hase and GTP cyclohyd olase II, bo h in he ibofla in ( i amin
567
B2) biosyn he ic pa hway, we e s ongly ep essed oge he wi h
568
3-phosphose ine amino ans e ase, which is linked o he syn he-
569
ses o glycine, se ine and h eonine bu also plays a ole in he bio-
570
syn hesis o py idoxine ( i amin B6). Ribofla in p o ec s cells om
571
oxida i e inju ies (Sugiyama, 1991; Pe umal e al., 2005), and MSB-
Fig. 3. Me abolic unc ion and schema ic cellula localiza ion o MSB-s ess- esponsi e A. nidulans p o eins. P o eins wi h pu a i e unc ions a e summa ized in Table 1 and
a e labeled he e wi h hei locus IDs. Symbols s,d,hand jindica e inc eased p o ein, dec eased p o ein, inc eased mRNA and dec eased mRNA le els, espec i ely (Table 1;
Supplemen a y 3;Pócsi e al., 2005). Ques ion ma ks e e o ‘ambi alen ’ changes in p o ein le els (Table 1;Fig. 2). Dashed lines indica e mul i-s ep me abolic pa hways.
Please no e he ema kably al e na ing p o ein induc ion and ep ession pa e n obse able in he glycoly ic me abolic pa hway be ween he AcuG and PkiA enzymes.
T. Pusz ahelyi e al./ Fungal Gene ics and Biology xxx (2010) xxx–xxx 9
YFGBI 2267 No. o Pages 12, Model 5G
30 Augus 2010
Please ci e his a icle in p ess as: Pusz ahelyi, T., e al. Compa ison o ansc ip ional and ansla ional changes caused by long- e m menadione exposu e
in Aspe gillus nidulans. Fungal Gene . Biol. (2010), doi:10.1016/j. gb.2010.08.006