ArrayMAPPIT: high-throughput interactome analysis in mammalian cells Cytokine Receptor Lab Ghent University - Dept. Medicine and Health Sciences VIB -

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

http://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


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


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


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


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


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


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

Documents

ArrayMAPPIT: high-throughput interactome analysis in mammalian cells Cytokine Receptor Lab Ghent University - Dept. Medicine and Health Sciences VIB -