Transcript patenten

Mapping the human interactome:
a
update
the genomic revolution in numbers
from gene sequence to protein function
large-scale protein interaction mapping
yeast two-hybrid
AP/MS
binary protein interactions
transient
protein complexes
stable
different network topology
>> complementary
different interactome subspace interrogated
similar high quality
from Yu et al. High-quality binary protein interaction map of the yeast interactome network. Science 2008
MAPPIT validation of Y2H protein network maps
yeast two-hybrid
x
y
DB
AD
x y
DB
AD
Pol
reporter gene
other two-hybrid methods
x
y
F
F’
x y
F F’
other two-hybrid methods
x
y
F
F’
x y
F F’
MAPPIT
cytokine
P
P
Jak
Jak
P
P
STAT
P
STAT
P
y x
x
P
P
STAT
STAT
y
Pol
reporter gene
• operates in mammalian cells
• ligand-inducible > extra level of control
• simple readout > automation
MAPPIT validation of Y2H protein network maps
>> CCSB-YI1: 1.809 interactions between 1.278 proteins
(estimated interactome size 18.000 +/- 4.500)
MAPPIT validation of Y2H protein network maps
WI-2007: 1.816 interactions between 1.496 proteins
(estimated interactome size 115.600 +- 26.400)
MAPPIT validation of Y2H protein network maps
~700 full length (bait) x ~700 fragments (prey)
40 fragments per ORF
>> 755 interactions between 522 proteins
(only 92 previously identified by Y2H !)
MAPPIT validation of Y2H protein network maps
framework for large-scale Y2H human interactome mapping
-validation of available HT-YTH interactome maps:
(Vidal & Wanker groups)
>> high quality (> literature curated)
-estimation of interactome size:
~130.000 interactions
MAPPIT validation of Y2H protein network maps
framework for large-scale Y2H human interactome mapping
-validation of available HT-YTH interactome maps:
(Vidal & Wanker groups)
>> high quality (> literature curated)
-estimation of interactome size:
~130.000 interactions
-standardized confidence scoring method
empirical confidence score
from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009
empirical confidence score
from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009
mapping the human interactome
•
3 year NIH grant
•
Y2H: 16.000 x 16.000 full lenght human ORFs (~ 50% of total matrix of 22.000 x 22.000)
•
interaction toolkit re-test: ~25-30.000 interactions (~10.000/year; ~20% of the map)
what did we learn ?
benchmarking binary interaction mapping methods
>> MAPPIT performance is similar to that of the other tested methods
from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009
benchmarking binary interaction mapping methods
>> the interaction mapping methods are highly complementary
from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009
the ORFeome collection
• 15.483 full length human ORFs
• derived from Mammalian Gene Collection (MGC)
• cloned in Gateway vectors
from http://horfdb.dfci.harvard.edu/
MAPPIT for large-scale interactome analysis ?
•
high quality assay
•
access to a large collection of easily transferred cDNAs
•
different and complementary network subspace probed
> screening for novel interactions
towards an efficient screening format: reverse transfection
nucleic acid
spot
transfection
reagent
add
cells
incubate
ArrayMAPPIT screening
human ORFeome collection
prey (+reporter)
plasmid
transfection
reagent
reverse
transfection mix
-/+ ligand
MAPPIT prey collection
MAPPIT bait
cell line
MAPPIT prey array
(stable for months !)
luciferase read-out
current screening setup
•
prey collection: 2.000 human ORF preys (GO annotation “signal transduction”)
•
assay format: 96well > 384well
•
automation:
– Tecan EVO150 (DNA preps)
– Tecan EVO200/Perkin-Elmer Envision (array production array + assay read-out)
screening for interaction partners of E3 ligase complex adaptors
“Specificity module”:
SCF – Skp1 + F-box protein
ECS – ElonginB/C + SOCS-box protein
SKP1 screen
100
FBXO46
FBXL8
FBXW11
FBXW9
FBXW11
FBXW9
BTRC
FBXO46
FBXL8
unstimulated
0,01
BTRC
10
1
0,1
1
10
0,01
stimulated
0,1
• 10-fold cut-off >> 5 hits: 3 known (blue), 3 novel (green); all F-box proteins
• no other known Skp1 interaction partners in the array
100
Elongin C screen
100
SOCS2
SPSB2
RAB40B
ASB9
ASB1
TCEB2
10
ASB6
ASB8
SPSB1
WSB1
ASB2
SPSB4
unstimulated
0,01
1
0,1
1
10
0,01
•
•
•
•
stimulated
0,1
10-fold cut-off >> 5 hits: 4 known and 1 novel (all SOCS-box proteins)
5-fold cut-off >> 8 additional hits: 4 known interactors (all SOCS-box proteins)
3-fold cut-off >> 14 additional hits: 2 known and 1 novel interactor (all SOCS-box proteins)
6 false negatives
100
Co-IP confirmation
SPSB4
SPSB2
mock
SOCS2
Elongin C
FBXO46
FBXW9
FBXW11
mock
SKP1
WB anti-E
lysate
WB anti-Elongin C
WB anti-E
IP anti-Flag
WB anti-Elongin C
WB anti-Flag
IP anti-Flag
WB anti-Flag
MAPPIT cDNA library screening
MACS
enrichment
anti-mIgG-PE
anti-hIL5R
hIL5R
mEcoR
5’LTR
CMV
LR-F3
bait
CD90
CMV
gp130
rPAP1
prey
hIL5RαΔcyt
FACS sort
anti-PE
magnetobead
hIL5R
retroviral
prey cDNA
library
SKP1 screen
Symbol
Description
FBXL8
FBXL15
FBXW5
FBX044
FBXO2
CDCA3
FBXL6
FBXW9
F-box and leucine-rich repeat protein 8
F-box and leucine-rich repeat protein 15
F-box and WD domain protein 5
F-box protein 44
F-box protein 2
cell division cycle associated 3
F-box and leucine-rich repeat protein 6
F-box and WD-40 domain protein 9
• 6 known SKP1 interacting proteins
• 2 novel interaction partners (both F-box proteins)
Number of clones
(fusions)
9 (5)
1 (1)
12 (5)
9 (7)
1 (1)
1 (1)
3 (1)
5 (3)
Array versus cDNA library screening
cDNA library screening
array screening
‘open’: large & diverse prey pool
‘closed’: fixed set of preys
labour intensive
fast
prey identification is tedious
position in array determines prey identity
MAPPIT for large-scale interactome analysis ?
•
high quality assay
•
access to a large collection of easily transferred cDNAs
•
different and complementary network subspace probed
> > screening for novel interactions
•
mammalian background
yeast two-hybrid interaction maps are static
•
the human interactome is not static but dynamic
– many protein-protein interactions are conditional or context-dependent
– require post-translational modifications and/or structural alterations
– require co-factors, adaptors or regulatory proteins
•
yeast cell doesn’t provide the normal cellular environment for human proteins
– no accessory proteins
– no modifications
– no context-dependent interactions
MAPPIT for large-scale interactome analysis ?
•
high quality assay
•
access to a large collection of easily transferred cDNAs
•
different and complementary network subspace probed
> > screening for novel interactions
•
mammalian background
> > mapping protein network dynamics
mapping dynamic aspects of protein networks ?
treatment B
-/+ ligand
treatment A
MAPPIT bait
cell line
treatment C
mapping dynamic aspects of protein interactions: GR signalling
cytoplasm
nucleus
NFkB
monomer
dimer
p53
MAPPIT can detect these changes in protein interactions
luciferase activity (fold induction)
30
25
unstimulated
stimulated
unstimulated + DEX
stimulated + DEX
20
15
10
5
0
GR bait
+ NS4A prey
GR bait
+ p53 prey
GR bait
+ Hsp90 prey
screening for DEX-dependent GR interactions
- DEX
-/+ ligand
GR-bait
expressing
cells
+ DEX
screening for DEX-dependent GR interactions
40
- dexamethasone
+ dexamethasone
luciferase values (fold induction)
35
30
25
20
15
10
5
0
GR bait
STOP
p53
Hsp90 FBXW9
HBP1
STAT4
SOCS3
NRIP1
FASTK
LPXN
NCOA4
SHC4
DOK3
PPP5C
STOP
p53
Hsp90 FBXW9
HBP1
STAT4
SOCS3
NRIP1
FASTK
LPXN
NCOA4
SHC4
DOK3
PPP5C
100
90
luciferase values (fold induction)
80
70
60
50
40
30
20
10
0
Skp1 bait
screening for DEX-dependent GR interactions
+ STAT3 – STAT5A – HGMB2
40
- dexamethasone
+ dexamethasone
35
luciferase values (fold induction)
6 stably interacting proteins:
STAT3, STAT5A, HGMB2 (known)
HBP1, STAT4, SOCS3
30
25
20
15
10
5
0
GR bait
STOP
p53
Hsp90 FBXW9
HBP1
STAT4
SOCS3
NRIP1
FASTK
LPXN
NCOA4
SHC4
DOK3
PPP5C
STOP
p53
Hsp90 FBXW9
HBP1
STAT4
SOCS3
NRIP1
FASTK
LPXN
NCOA4
SHC4
DOK3
PPP5C
100
90
luciferase values (fold induction)
80
70
60
50
40
30
20
10
0
Skp1 bait
screening for DEX-dependent GR interactions
+ STAT3 – STAT5A – HGMB2
40
- dexamethasone
+ dexamethasone
35
luciferase values (fold induction)
6 DEX-inducible interactions:
NRIP1 (known interactor)
NCOA4 (AR interactor)
FASTK, LPXN, SHC4, DOK3
30
25
20
15
10
5
0
GR bait
STOP
p53
Hsp90 FBXW9
HBP1
STAT4
SOCS3
NRIP1
FASTK
LPXN
NCOA4
SHC4
DOK3
PPP5C
STOP
p53
Hsp90 FBXW9
HBP1
STAT4
SOCS3
NRIP1
FASTK
LPXN
NCOA4
SHC4
DOK3
PPP5C
100
90
luciferase values (fold induction)
80
70
60
50
40
30
20
10
0
Skp1 bait
screening for DEX-dependent GR interactions
+ STAT3 – STAT5A – HGMB2
40
- dexamethasone
+ dexamethasone
35
luciferase values (fold induction)
1 DEX-repressible interaction:
PPP5C (known interactor)
30
25
20
15
10
5
0
GR bait
STOP
p53
Hsp90 FBXW9
HBP1
STAT4
SOCS3
NRIP1
FASTK
LPXN
NCOA4
SHC4
DOK3
PPP5C
STOP
p53
Hsp90 FBXW9
HBP1
STAT4
SOCS3
NRIP1
FASTK
LPXN
NCOA4
SHC4
DOK3
PPP5C
100
90
luciferase values (fold induction)
80
70
60
50
40
30
20
10
0
Skp1 bait
screening for DEX-dependent GR interactions
ArrayMAPPIT - further development
•
prey collection: 2.000 human ORF preys > 10.000 (end 09)
•
assay format: 384well > glass slides (?)
•
increase assay sensitivity – decrease assay variability
•
data-management, optimized experimental setup, objective scoring and quality control
tracking (StatGent)
CRL
CCSB
Jan Tavernier
Marc Vidal
Dominiek Catteeuw
Els Pattyn
Delphine Lavens
Leentje De Ceuninck
Isabel Uyttendaele
Celia Bovijn
Laura Icardi
Margarida Maia
Sylvie Seeuws
Lennart Zabeau
Irma Lemmens
Anne-Sophie De Smet
Elien Ruyssinck
Viola Gesellchen
Tim Van Acker
Frank Peelman
Julie Piessevaux
Peter Ulrichts
Annick Verhee
Joris Wauman
José Van der Heyden
Nele Vanderroost
Dieter Defever
& co