MAPPIT Analysing Protein Interactions in Human Cells VIB/UGent Workshop October 2009
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Transcript of MAPPIT Analysing Protein Interactions in Human Cells VIB/UGent Workshop October 2009
MAPPIT Analysing Protein Interactions in Human Cells
VIB/UGent Workshop October 2009
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
P YPYP
YPYP
STAT responsive promoter
cytoplasm
nucleus
JAK-STAT signalling
JAK2 JAK2
F
F
F
F
F
F
Y
Y
cytokine receptor
leptin receptor (Y>F)
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
bait
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
YYYY
prey
gp130
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
STAT3
Y
Y
MAPPIT
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
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
Y PYP
reporter generPAP1 promoter Y PYP
MAPPIT
MAPPIT example
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
160.0
pSEL1-EpoR +pMG1-CIS
pSEL1-EpoR +pMG1-SVT
pSEL1-EpoR +pMET7-CIS
pSEL1-EpoRY-F +pMG1-CIS
pSEL1-p53 + pMG1-CIS
NS Epo
Epo
JAK2
F
F
F
Y
EpoR
JAK2
F
F
F
Y
EpoR
P
P
Y YP P
YY
YY
CIS
Montoye et al., Blood 105, 4264-4271 (2005)Lavens et al., J. Cell Sci. 119, 2214-2224 (2006)Montoye et al., FEBS Lett. 580, 3301-3307 (2006)Piessevaux et al., J. Biol. Chem. 281, 32953-66 (2006)Lavens et al., Biochem. J., 401 257-267 (2007)Ulrichts et al., FEBS Lett. 581, 629-636 (2007) Erkeland et al., Oncogene 26, 1984-1995 (2007)Uyttendaele et al., Mol. Endocrinol. 21, 2821-2831 (2007)Wauman et al., Mol. Endocrinol. 22, 965-977 (2008)Ulrichts et al., Immunol. Lett. 115, 141-148 (2008)Piessevaux et al., J. Biol. Chem. 283, 21334-21346 (2008)Pattyn et al., J. Virol. Methods 153, 7-15 (2008)Lemmens et al., Biochem Soc. Trans 36, 1448-1451 (2008)Piessevaux et al., Cytokine Growth Factor Rev. 19, 371-381 (2008)Lievens et al., J. Proteome Res. 8, 877-886 (2009)Ulrichts et al., Meth. Mol. Biol., 517, 133-144 (2009).Lievens et al., TiBS, in press.De Ceuninck et al., J. Biol. Chem., submittedUlrichts et al.,J. Cell Sci., submitted.Lavens et al., PLoS Pathogens, submitted.
MAPPIT analysis of HIV Reverse Transcriptase interactions
1
15
3
110
153
47
0
50
100
150
200gp
130-
SV
T
gp13
0-TR
IP13
gp13
0-R
Tp51
gp13
0-R
Tp66
gp13
0-R
Tp51
gp13
0-R
Tp66
RTp51
fold
indu
ctio
n
FLAG-gp130-RTp66
FLAG-gp130-RTp51
p51/p66=0.3µM p66/p66=4.0µM p51/p51=230µM
RTp51 bait RTp66 bait
MAPPIT as a tool for drug screening
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
RTp66b +RTp51p
RTp66b +RTp66p
RTp51b +RTp51p
RTp66b +RTp51p
RTp66b +RTp66p
RTp51b +RTp51p
RTp66b +RTp51p
RTp66b +RTp66p
RTp51b +RTp51p
NC efavirenz 0.5µM efavirenz 5µM
luci
fera
se c
ps
NC
Epo
Efavirenz stabilizes the HIV RT dimer
Interactomes
• Large-scale programs to map complete interactomes of an organism collaboration with Marc Vidal group (DFCI, Harvard U, Boston):
systematic matrix-based Y2H analysis
Network quality : coverage (false negatives)accuracy (false positives)
use of MAPPIT as an orthogonal assay
Interactomes
MAPPIT-based verification of Y2H interactome datasets:• Gateway-mediated transfer of ORF’s to MAPPIT vectors• Systematic MAPPIT analysis in 384-well format high-confidence maps
Yu et al., High quality binary protein interaction map of the yeast interactome networkScience (2008)
Simonis et al., Empirically-controlled mapping of the C. elegans protein-protein interactome network
Nat. Methods (2009)Venkatesan et al., An empirical framework for binary interactome mapping,
Nat. Methods (2009)
The data quality of high-throughput Y2H-based interactome datasets is high Low coverage and detectability accounts for the low overlap between different
large-scale datasets The estimated size of the human interactome is around 130.000 binary interactions,
current coverage is 8%
MAPPIT performance
0
5
10
15
20
25
30
35
LUMIER MAPPIT Y2H PCA wNAPPA
% GSP scored positive% GSN scored positive
Braun et al., An experimentally derived confidence score for binary protein-protein interactions. Nat. Methods (2009)
Comparison of interactomes
Yu et al., Science, 2008
ArrayMAPPIT screening assay
MAPPIT prey collection
prey plasmid(+reporter)
transfectionreagent
reverse transfection mix
MAPPIT prey array
luciferase read-out MAPPIT baitcell line
activation
human ORFeome collection
10-fold induction
0,01
0,1
1
10
100
0,01 0,1 1 10 100
unstimulated
stim
ula
ted
FBXO46
FBXW9
BTRC
FBXW11
BTRCFBXW9
FBXW11
FBXO46
FBXL8
FBXL8
L
J AK2
F
F
F
Y
SKP1
J AK2
F
F
F
Y
SKP1
P
P
P
P
YY
YY
?
ArrayMAPPIT screening: Skp1 interactions
Lievens et al., J. Proteome Res. 2009.
10-fold induction
5-fold induction
3-fold induction
0,01
0,1
1
10
100
0,01 0,1 1 10 100
unstimulated
stim
ula
ted
SOCS2
SPSB2
SPSB4
SPSB1
ASB8
ASB9
ASB1
ASB2WSB1
ASB6TCEB2
RAB40B
L
J AK2
F
F
F
Y
EloC
J AK2
F
F
F
Y
EloC
P
P
P
P
ArrayMAPPIT screening: EloC interactions
ArrayMAPPIT – towards the dynamics of PPI networks
treatment A
treatment B
treatment C
ligand
JAK2
F
F
F
Y
GR
JAK2
F
F
F
Y
GR
P
P
ArrayMAPPIT – Glucocorticoid Receptor interactions
ligand
JAK2
F
F
F
Y
GR
JAK2
F
F
F
Y
GR
P
P
ArrayMAPPIT – Glucocorticoid Receptor interactions
YY
YY
Hsp90
-Dex
ligand
JAK2
F
F
F
Y
GR
JAK2
F
F
F
Y
GR
P
P
ArrayMAPPIT – Glucocorticoid Receptor interactions
YY
YY
p53
+Dex
YY
YY
Hsp90
GR bait
Skp1 bait
ArrayMAPPIT – Glucocorticoid Receptor interactions
GR bait
Skp1 bait
ArrayMAPPIT – Glucocorticoid Receptor interactions
GR bait
Skp1 bait
ArrayMAPPIT – Glucocorticoid Receptor interactions
GR bait
Skp1 bait
ArrayMAPPIT – Glucocorticoid Receptor interactions
Lievens et al. (2004) Methods Mol. Biol.
lacZeoRFRTSV
hIL5Rα-ΔcytrPAP1
mEcoR
Hek293 Flp-In T-Rex 44 cells
FACS-based MAPPIT screening
FACS-based MAPPIT screening
baitEpoR/LR-LRF3CMV
JAK2
JAK2
FF
F
FF
FY
bait
bait
Y
hIL5Rα-ΔcytrPAP1
mEcoR
Lievens et al. (2004) Methods Mol. Biol.
FACS-based MAPPIT screening
baitEpoR/LR-LRF3CMV
JAK2
JAK2
FF
F
FF
FY
bait
bait
Y
ypreygp1305’LTR
YYYY
prey
YYYY
prey
hIL5Rα-ΔcytrPAP1
retroviral prey cDNA library
mEcoR
Lievens et al. (2004) Methods Mol. Biol.
FACS-based MAPPIT screening
baitEpoR/LR-LRF3CMV
JAK2
JAK2
FF
F
FF
FY
bait
bait
Y
ypreygp1305’LTR
YYYY
prey
YYYY
prey
ligand
JAK2F
F
F
Y
bait
JAK2F
F
F
Y
bait
P
P
ligandligand
ligandligand
YY
YY
prey
hIL5Rα-ΔcytrPAP1
mEcoR
Lievens et al. (2004) Methods Mol. Biol.
FACS-based MAPPIT screening
baitEpoR/LR-LRF3CMV
JAK2
JAK2
FF
F
FF
FY
bait
bait
Y
ypreygp1305’LTR
YYYY
prey
YYYY
prey
ligand
JAK2F
F
F
Y
bait
JAK2F
F
F
Y
bait
P
P
ligandligand
ligandligand
YY
YY
prey
PP
PP
hIL5Rα-ΔcytrPAP1
hIL5
Rα
-Δcy
tmEcoR
Lievens et al. (2004) Methods Mol. Biol.
FACS-based MAPPIT screening
Yanti-hIL5Rα
Y
Y
anti-PEmagnetobead
MACS presort(enrich-deplete-enrich)
baitEpoR/LR-LRF3CMV
JAK2
JAK2
FF
F
FF
FY
bait
bait
Y
ypreygp1305’LTR
YYYY
prey
YYYY
prey
ligand
JAK2F
F
F
Y
bait
JAK2F
F
F
Y
bait
P
P
ligandligand
ligandligand
YY
YY
prey
PP
PP
hIL5Rα-ΔcytrPAP1
hIL5
Rα
-Δcy
t
Yanti-mIgG-PE
mEcoR
Lievens et al. (2004) Methods Mol. Biol.
FACS-based MAPPIT screening
Yanti-hIL5Rα
Y
Y
anti-PEmagnetobead
baitEpoR/LR-LRF3CMV
JAK2
JAK2
FF
F
FF
FY
bait
bait
Y
ypreygp1305’LTR
YYYY
prey
YYYY
prey
ligand
JAK2F
F
F
Y
bait
JAK2F
F
F
Y
bait
P
P
ligandligand
ligandligand
YY
YY
prey
PP
PP
hIL5Rα-ΔcytrPAP1
hIL5
Rα
-Δcy
t
Yanti-mIgG-PE
anti-hIL5RαhIL5
Rα-Δ
cyt
anti-mIgG-PE
FACS single cell
sort
mEcoR
YY
Lievens et al. (2004) Methods Mol. Biol.
MAPPIT cDNA library screening assay
hSkp1A-bait versus Hek293T prey cDNA library
ligand
JAK2F
F
F
YJAK2
F
F
F
Y
P
P
Skp1ASkp1A
# clones # fusionsknown interactors
1 FBXL8 - F-box and leucine-rich repeat protein 8 6 42 FBXL15 - F-box and leucine-rich repeat protein 15 1 13 FBXW5 - F-box and WD domain protein 5 6 44 FBX044 - F-box protein 44 6 55 FBXO2 - F-box protein 2 1 16 CDCA3 - cell division cycle associated 3 1 1
related putative interactors1 FBXL6 - F-box and leucine-rich repeat protein 6 3 12 FBXW9 - F-box and WD-40 domain protein 9 3 3
SUBTOTAL 27 20
other putative interactors1 CHCHD2 - Coiled coil helix coiled coil helix domain containing 2 2 22 CCDC95 - coiled-coil domain containing 95 1 13 H2AFZ - H2A histone family, member Z 2 24 DDX11 - DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 11 1 15 MAZ - MYC-associated zinc finger protein 1 16 RPS2 - ribosomal protein S2 5 37 NOLA1 - nucleolar protein family A, member 1 1 18 TTC9C - tetratricopeptide repeat domain 9C 4 49 PDIA6 - protein disulfide isomerase family A, member 6 1 1
10 PIP5K2B - phosphatidylinositol-5-phosphate 4-kinase, type II beta 1 111 HCFC1 - host cell factor C1 2 112 NAPA - N-ethylmaleimide-sensitive factor attachment protein, alpha 1 113 RAVER1 - ribonucleoprotein, PTB-binding 1 1 114 MRFAP1 - Mof4 family associated protein 1 1 115 ID3 - inhibitor of DNA binding 3 2 116 ATP5G2 - ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C2 (subunit 9) 1 117 PPIE - peptidylprolyl isomerase E 1 118 FTL - ferritin light chain 1 119 TPD52L1 - tumor protein D52-like 1 1 120 ARF1 - ADP-ribosylation factor 1 1 121 Hypothetical protein DKFZP434H132 1 122 ERH - enhancer of rudimentary homolog (Drosophila) 1 123 CKB - creatine kinase brain type 1 1
SUBTOTAL 34 30
TOTAL 61 50
YY
YY
?
Lievens et al., J. Proteome Res., 2009.
MAPPIT summary
• Operates in intact human cells = close to normal physiological context
• Easy to perform, simple readout
• High sensitivity and specificityTight background control
interaction and effector zone are separated ligand-inducible system
• Can detect weak/transient interactions indirect interactions
modification-dependent interactions (Heteromeric MAPPIT)
• High-throughput modus ArrayMAPPIT runs on 96 and 384 well format on Tecan Evo robotics platform
Applications in interactome mapping (20.000 PPI’s/year) and drug screening cDNA library screens
Limitations of MAPPIT
• No real-time measurements FRET/BRET
• Subcellular localization Designed for cytosolic proteins
• Difficult for membrane proteins (e.g. GPCR’s, Ion Channels) Split Ubiquitin Y2H
MAPPIT summary
• Operates in intact human cells = close to normal physiological context
• Easy to perform, simple readout
• High sensitivity and specificityTight background control
interaction and effector zone are separated ligand-inducible systemSubcellular localization
• Can detect weak/transient interactions indirect interactions
modification-dependent interactions (Heteromeric MAPPIT)
• High-throughput modus ArrayMAPPIT runs on 384 well format on Tecan Evo robotics platform
Applications in large-scale interactome mapping (20.000 PPI’s/year) cDNA library screens
Inducible MAPPIT: transient transfections
Best configuration: pTREtight-prey (Clontech): inducible prey rtTA-Advanced (Clontech)
No preformed bait/prey complexes Can the protein folding pathway be targeted by small molecules?
Inducible mappit: transient Vif-ElonginC interaction
inducible mappit
21
23 23
0
5
10
15
20
25
30
35
Epo Dox Dox+Epo Dox + Epo after 6hr
prey=pTRE-tight G2-elongin C (0,5µg) bait=pCEL-VIF (0,5µg)
fold
in
du
ctio
n
+ Epo
Dox 1000 ng/ml + Epo
Dox 100 ng/ml + Epo
Dox 10 ng/ml + Epo
1 2 3 41.
2.
3.
4.
WB anti-flag
WB anti-actin
0
5
10
15
20
25
prey: pTRE tight-G2-elonginC bait: pCEL-VIF
fold
indu
ctio
n
www.mappit.be