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

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


YY

YY

Hsp90

-Dex

ligand

JAK2

F

F

F

Y

GR

JAK2

F

F

F

Y

GR

P

P


YY

YY

p53

+Dex

YY

YY

Hsp90

GR bait

Skp1 bait


Lievens et al. (2004) Methods Mol. Biol.

lacZeoRFRTSV

hIL5Rα-ΔcytrPAP1

mEcoR

Hek293 Flp-In T-Rex 44 cells

FACS-based MAPPIT screening


baitEpoR/LR-LRF3CMV

JAK2

JAK2

FF

F

FF

FY

bait

bait

Y

hIL5Rα-ΔcytrPAP1

mEcoR



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



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



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



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



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


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

mappit analysing protein interactions in human cells vib/ugent workshop october 2009

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