Post on 20-Dec-2015
ArrayMAPPIT: high-throughput interactome analysis in mammalian cells
Cytokine Receptor Lab
Ghent University - Dept. Medicine and Health Sciences VIB - Dept. Medical Protein Research
Belgium
www.crl-mappit.be
• MAmmalian Protein-Protein Interaction Trap
• mammalian two-hybrid based on functional complementation of the JAK-STAT signalling pathway
The MAPPIT toolbox
• MAPPIT (2H): protein-protein interaction
• ReverseMAPPIT (R2H): disruption of protein-protein interaction
• MASPIT (3H): compound-protein interaction
JAK JAK
type I cytokine receptor
Y
Y
Y
Y
Y
Y
Y
YJAK kinase
extracellular
intracellular
JAK-STAT signalling
Y
Y
Y
Y
Y
Y
JAKY
JAKY
cytokine
P
P
JAK-STAT signalling
Y
Y
Y
Y
Y
Y
JAKY
JAKY
P
P
P
P
P
P
P
P
JAK-STAT signalling
Y
Y
Y
Y
Y
Y
JAKY
JAKY
P
P
P
P
P
P
P
P
Y
Y
STAT
JAK-STAT signalling
Y
Y
Y
Y
Y
Y
JAKY
JAKY
P
P
P
P
P
P
P
PYP
YP
YPYP
STAT responsive promoter
cytoplasm
nucleus
JAK-STAT signalling
JAK2 JAK2
F
F
F
F
F
F
Y
Y
Eyckerman et al. (2001) Nat. Cell Biol.
cytokine receptor
leptin receptor (Y>F)
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
MAPPIT
Eyckerman et al. (2001) Nat. Cell Biol.
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
MAPPIT
Eyckerman et al. (2001) Nat. Cell Biol.
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
bait
MAPPIT
Eyckerman et al. (2001) Nat. Cell Biol.
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
YYYY
prey
gp130
MAPPIT
Eyckerman et al. (2001) Nat. Cell Biol.
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
MAPPIT
Eyckerman et al. (2001) Nat. Cell Biol.
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
STAT3
Y
Y
MAPPIT
Eyckerman et al. (2001) Nat. Cell Biol.
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
Y
Y
reporter generPAP1 promoter
P
P
MAPPIT
Y PYP
Eyckerman et al. (2001) Nat. Cell Biol.
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
Y PYP
reporter generPAP1 promoter Y PYP
MAPPIT
Eyckerman et al. (2001) Nat. Cell Biol.
features
• operates in intact human cells = close to normal physiological context
• works in different cell types (epithelial, haematopoietic, neuronal)
• tight background control– interaction and effector zone are separated– ligand-inducible system
• easy to perform, simple readout, can be automated
pathway analysis of early steps in Toll-Like Receptor signalling
TLR signaling: TLR4 dimerisation
*
0
5
10
15
20
25
30
SVT-prey TLR4ic-prey
fold
indu
ctio
n p
TLR4ic-bait
TLR signaling: TLR4 adaptor interactions
0
5
10
15
20
25
30
SVT-prey Tram-prey Mal-prey
fold
indu
ctio
n
TLR4ic-bait* *
TLR signaling: Mal-MyD88 homo- and heterodimerisation
0
5
10
15
20
25
30
35
SVT-prey MyD88-prey Mal-prey
fold
indu
ctio
n
Mal-bait
MyD88-bait
*
TLR signaling: Mal bridges between MyD88 and TLR4
0
5
10
15
20
25
SVT-prey MyD88-prey wt mal + myd88-prey
fold
indu
ctio
n
TLR4ic-bait
*
0
5
10
15
20
25
SVT-prey MyD88-prey wt mal + myd88-prey
fold
indu
ctio
n
TLR4ic-bait
TLR signaling: MyD88 associates with IRAK-4
0
2
4
6
8
10
12
14
16
18
SVT-prey MyD88-prey
fold
indu
ctio
n
IRAK-4 -bait
0
5
10
15
20
25
SVT-prey IRAK4-prey
fold
indu
ctio
n
MyD88-bait
*
TLR signaling: MyD88 bridges between MAL and IRAK-4
0
2
4
6
8
10
12
14
16
SVT-prey Mal-prey MyD88+ Mal-prey
fold
ind
uct
ion
IRAK-4 -bait
*
0
2
4
6
8
10
12
14
16
SVT-prey Mal-prey MyD88+ Mal-prey
fold
ind
uct
ion
IRAK-4 -bait
TLR signaling: IRAK-4 interacts with IRAK-1
*0
1
2
3
4
5
6
SVT-prey IRAK1-prey
fold
indu
ctio
n
p
IRAK4-bait
pathway analysis
• toll-like receptor• leptin receptor• erythropoietin receptor• growth hormone receptor• granulocyte colony-stimulating factor receptor
Lemmens et al., Nucleic Acids Res. 2003
Montoye et al., Blood 2005
Lavens et al., J. Cell Sci. 2006
Montoye et al., FEBS Lett. 2006
Piessevaux et al., J. Biol. Chem. 2006
Lavens et al., Biochem. J. 2007
Ulrichts et al., FEBS Lett. 2007
Erkeland et al., Oncogene 2007
Uyttendaele et al., Mol. Endocrinol. 2007
Wauman et al., Mol. Endocrinol. 2008
Piessevaux et al., J. Biol. Chem. 2008
Ulrichts et al., Immunol. Lett. 2008
validation of Y2H interactome maps
• Boxem et al. A protein domain-based interactome network for C. elegans early embryogenesis, Cell 2008
• Yu et al. High accuracy high throughput binary interactome datasets for S. cerevisiae, Science 2008
• Braun et al. An experimentally derived confidence score for binary protein-protein interactions, Nat. Methods, under revision
• Simonis et al. A high confidence C. elegans protein-protein interaction map, Nat. Methods., under revision
• Venkatesan et al. Empirical sizing of the human interactome by self-controlled binary mapping, Nat. Methods, under revision
% PRS positive
% RRS positive
% PRS positive
% RRS positive
% PRS positive
% RRS positive
performance
Braun et al.
performance
Braun et al.
ArrayMAPPIT screening assay
MAPPIT prey collection
prey (+reporter)plasmid
transfectionreagent
reverse transfection mix
MAPPIT prey array
luciferase read-out MAPPIT baitcell line
-/+ ligand
human ORFeome collection
screening for Skp1 interaction partners
SCF E3 ligase
screening for Skp1 interaction partners
1904 full length human ORFsfrom Vidal collection
(GO ‘signal transduction’)
luciferase read-out(average of duplicatesnormalized for plate
median)
LR(F3)-Skp1 baitexpressing cell line
-/+ leptin
screening for Skp1 interaction partners
10-fold induction
0,01
0,1
1
10
100
0,01 0,1 1 10 100
unstimulated
stim
ulat
ed
FBXO46
FBXW9
FBXL8
BTRC
FBXW11
comparing the effect of different treatments
treatment A
treatment B
treatment C
assaying the effect of exogenous treatments on PPIs
examples:• p53 interactions – nutlin3• HIV Reverse Transcriptase dimerisation - efavirenz• Glucocorticoid Receptor interactions - dexamethasone
Differential modulation of p53-mdm2/4 interactions
p53mdm2
p53mdm4
p53mdm4
p53mdm2
Nutlin
Differential modulation of p53-mdm2/4 interactions
0
5
10
15
20
25
30
p53 bait+ mdm2 prey
p53 bait+ mdm4 prey
luci
fera
se a
ctivi
ty (f
old
indu
ction
)
unstimulatedstimulatedunstimulated + nutlin3stimulated + nutlin3
0
5
10
15
20
25
30
p53 bait+ mdm2 prey
p53 bait+ mdm4 prey
luci
fera
se a
ctivi
ty (f
old
indu
ction
)
unstimulatedstimulatedunstimulated + nutlin3stimulated + nutlin3
HIV Reverse Transcriptase heterodimer
p66
p51
NNRTI binding site(allosteric)
NRTI binding site(polymerase active site)
analysis of RT dimerization
0
50
100
150
200
SVT
prey
RTp6
6pr
ey
RTp5
1pr
ey
SVT
prey
RTp6
6pr
ey
RTp5
1pr
ey
RTp66 bait RTp51 bait
fold
indu
ction
screen for HIV RT(p66) binders
0,01
0,1
1
10
100
0,01 0,1 1 10 100
unstimulated
stim
ulat
ed
5-fold induction
RT(p66)
RT(p51)
competition with wild type RT subunits
0
10
20
30
irrel
p66
p51
irrel
p66
p51
Bait: p66 Prey: p51 Bait: p51 Prey: p66
fold
indu
ction
stabilization of RT dimerisation by efavirenz
Kd (µM) 0.3 0.01 4 0.08 230 0.4
µM efavirenz
dose-reponse curve of efavirenz stabilizing effect
RTp66 bait + RTp51 prey
0
100
200
300
400
500
600
0,1 1 10 100 1000 10000
nM efavirenz
% o
f va
lue
wit
ho
ut
efa
vir
enz
NC
Epo
Glucocorticoid receptor signalling
p53
DEX dependent interactions with the GR
0
5
10
15
20
25
30
GR bait+ NS4A prey
GR bait+ p53 prey
GR bait+ Hsp90 prey
luci
fera
se a
ctivi
ty (f
old
indu
ction
)unstimulatedstimulatedunstimulated + DEXstimulated + DEX
0
5
10
15
20
25
30
GR bait+ NS4A prey
GR bait+ p53 prey
GR bait+ Hsp90 prey
luci
fera
se a
ctivi
ty (f
old
indu
ction
)unstimulatedstimulatedunstimulated + DEXstimulated + DEX
0
5
10
15
20
25
30
GR bait+ NS4A prey
GR bait+ p53 prey
GR bait+ Hsp90 prey
luci
fera
se a
ctivi
ty (f
old
indu
ction
)unstimulatedstimulatedunstimulated + DEXstimulated + DEX
prospects
• sensitive assay• physiologically relevant background• large prey collection• automated screening
>> analysis of the dynamics of interaction patterns in response to changes in intra-/extracellular environment
CRL
Jan Tavernier
Dominiek CatteeuwEls PattynDelphine LavensLeentje De CeuninckIsabel UyttendaeleCelia BovijnLaura IcardiMargarida MaiaSylvie SeeuwsLennart ZabeauIrma LemmensAnne-Sophie De SmetFrank PeelmanJulie PiessevauxPeter UlrichtsJosé Van der Heyden Nele VanderroostAnnick VerheeJoris Wauman
CCSB
Marc Vidal & co