Mapping the human interactome: a update. the genomic revolution in numbers

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

binary protein interactionstransient

AP/MS

protein complexesstable

different network topology

different interactome subspace interrogated

from Yu et al. High-quality binary protein interaction map of the yeast interactome network. Science 2008

>> complementary

similar high quality

MAPPIT validation of Y2H protein network maps

yeast two-hybrid

reporter genePol

x y

AD

x

DB

y

AD

DB

other two-hybrid methods

x

F

y

F’

x y

F’F

other two-hybrid methods

x

F

y

F’

x y

F’F

MAPPIT

reporter genePol

Jak

P

P y x

P

STATP

STATP

JakP

x

STAT

P

STATP

y

cytokine

• operates in mammalian cells

• ligand-inducible > extra level of control

• simple readout > automation


>> CCSB-YI1: 1.809 interactions between 1.278 proteins(estimated interactome size 18.000 +/- 4.500)


WI-2007: 1.816 interactions between 1.496 proteins(estimated interactome size 115.600 +- 26.400)


~700 full length (bait) x ~700 fragments (prey)

40 fragments per ORF

>> 755 interactions between 522 proteins

(only 92 previously identified by Y2H !)


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


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


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


benchmarking binary interaction mapping methods


>> the interaction mapping methods are highly complementary

the ORFeome collection

from http://horfdb.dfci.harvard.edu/

• 15.483 full length human ORFs

• derived from Mammalian Gene Collection (MGC)

• cloned in Gateway vectors

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

transfectionreagent

spotaddcells incubate

ArrayMAPPIT screening

MAPPIT prey collection

prey (+reporter)plasmid transfection

reagent

reverse transfection mix

MAPPIT prey array(stable for months !)

luciferase read-out MAPPIT baitcell line

-/+ ligand

human ORFeome collection

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

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

• 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

Elongin C screen

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

• 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

Co-IP confirmation

WB anti-E

WB anti-E

WB anti-Flag

mo

ck

lysate

IP anti-Flag

IP anti-Flag

WB anti-Elongin C

WB anti-Elongin C

WB anti-Flag

FB

XW

11

FB

XW

9

FB

XO

46

mo

ck

SO

CS

2

SP

SB

2

SP

SB

4

SKP1 Elongin C

hIL5Rα

Y

anti-hIL5Rα

Y

Y

anti-PEmagnetobead

baitLR-F3CMV hIL5RαΔcytrPAP1

hIL5Rα

Yanti-mIgG-PE FACS sort

mEcoR

YY

MACS enrichment

preygp1305’LTR CMVCD90

retroviral prey cDNA

library

MAPPIT cDNA library screening

SKP1 screen

Symbol Description Number of clones (fusions)

FBXL8 F-box and leucine-rich repeat protein 8 9 (5)FBXL15 F-box and leucine-rich repeat protein 15 1 (1)FBXW5 F-box and WD domain protein 5 12 (5)FBX044 F-box protein 44 9 (7)FBXO2 F-box protein 2 1 (1)CDCA3 cell division cycle associated 3 1 (1)FBXL6 F-box and leucine-rich repeat protein 6 3 (1)FBXW9 F-box and WD-40 domain protein 9 5 (3)

• 6 known SKP1 interacting proteins

• 2 novel interaction partners (both F-box proteins)

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





> > 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





> > screening for novel interactions

• mammalian background

> > mapping protein network dynamics

mapping dynamic aspects of protein networks ?

treatment A

treatment B

treatment C MAPPIT baitcell line

-/+ ligand

mapping dynamic aspects of protein interactions: GR signalling

p53

monomer

dimer

NFkB

cytoplasm nucleus

MAPPIT can detect these changes in protein interactions

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


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


screening for DEX-dependent GR interactions

- DEX

+ DEX GR-baitexpressing

cells

-/+ ligand


0

5

10

15

20

25

30

35

40

STOP p53 Hsp90 FBXW9 HBP1 STAT4 SOCS3 NRIP1 FASTK LPXN NCOA4 SHC4 DOK3 PPP5C

luci

fera

se v

alue

s (f

old

indu

ction

)

- dexamethasone

+ dexamethasone

0

10

20

30

40

50

60

70

80

90

100


luci

fera

se v

alue

s (f

old

indu

ction

)

GR bait

Skp1 bait


0

5

10

15

20

25

30

35

40


luci

fera

se v

alue

s (f

old

indu

ction

)

- dexamethasone

+ dexamethasone

0

10

20

30

40

50

60

70

80

90

100


luci

fera

se v

alue

s (f

old

indu

ction

)

GR bait

Skp1 bait

+ STAT3 – STAT5A – HGMB2

6 stably interacting proteins:

STAT3, STAT5A, HGMB2 (known)

HBP1, STAT4, SOCS3


0

5

10

15

20

25

30

35

40


luci

fera

se v

alue

s (f

old

indu

ction

)

- dexamethasone

+ dexamethasone

0

10

20

30

40

50

60

70

80

90

100


luci

fera

se v

alue

s (f

old

indu

ction

)

GR bait

Skp1 bait


6 DEX-inducible interactions:

NRIP1 (known interactor)

NCOA4 (AR interactor)

FASTK, LPXN, SHC4, DOK3


0

5

10

15

20

25

30

35

40


luci

fera

se v

alue

s (f

old

indu

ction

)

- dexamethasone

+ dexamethasone

0

10

20

30

40

50

60

70

80

90

100


luci

fera

se v

alue

s (f

old

indu

ction

)

GR bait

Skp1 bait


1 DEX-repressible interaction:

PPP5C (known interactor)


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

Jan Tavernier

Dominiek CatteeuwEls PattynDelphine LavensLeentje De CeuninckIsabel UyttendaeleCelia BovijnLaura IcardiMargarida MaiaSylvie SeeuwsLennart ZabeauIrma LemmensAnne-Sophie De SmetElien RuyssinckViola GesellchenTim Van AckerFrank PeelmanJulie PiessevauxPeter UlrichtsAnnick VerheeJoris WaumanJosé Van der Heyden Nele VanderroostDieter Defever

CCSB

Marc Vidal

& co

Documents

Mapping the human interactome: a update. the genomic revolution in numbers