Protein networks, Protein networks, moonlighting and moonlighting and

fuzzinessfuzziness

Peter Tompa

Institute of EnzymologyHungarian Academy of Sciences

Budapest, Hungary

Science of networks

- social networks

- transportation networks

- internet

- feeding networks

- neuronal networks

- intracellular networks

- metabolic networks

- expression networks

- protein-protein interactions (interactome)

The TAP tag The TAP tag methodmethod

Gene-specific casette

containing TAP tag by PCR

Insert at 3’ end of ORF by homologous recombination

Extract of recombinant cells

Tandem affinity purification (TAP)

Analysis (SDS-PAGE, MS)

Rigaut et al. (1999) Nature Biotech. 17, 1030

YeastYeast twotwo-h-hyybrid brid methodmethod (Y2H)(Y2H)

Fields and Song (1989) Nature 340, 245

bait

prey

The yeast interactomeThe yeast interactome

node

edge

hub

Uetz et al. (2000) Nature 403, 623

Comparison of Comparison of TAP-tag TAP-tag andand Y2H Y2H

- real complexes

- tag interference

- strength of interaction

- stoichiometry

- one condition

- pairwise interactions

- no posttransl. mod.

- third partner?

- bait-prey nonprod.

- compartmentalisation

TAP-tag Y2H

- comparison?

- partial coverage!

How to relate Y2H and TAP-tag How to relate Y2H and TAP-tag data?data?

A

ED

B

C

G

F L

H

JKI

TAP

A B

A C

A E

A H

x

tagY2H

x

““spoke” model better than spoke” model better than “matrix”“matrix”

A

ED

B

C

G

F L

H

JKI

TAPtag spoke (pairwise

interactions of bait):

a-b, a-c, a-d, a-e

matrix (pairwise interactions of all components):

a-b, a-c, a-d, a-e, b-c, b-d, b-e, c-d, c-e, d-e

Bader (2002) Nat. Biotech. 20, 991

Deane et al. (2002) MCP 1, 349

Partial coverage (Y2H yeast Partial coverage (Y2H yeast data)data)

Partial coverage (HTS vs. low-Partial coverage (HTS vs. low-throughput)throughput)

Gandhi (2006) Nat. Gen. 38, 285

Partial coverage (all Partial coverage (all interactions)interactions)

Gandhi (2006) Nat. Gen. 38, 285

Human 25.464

Yeast 16.069

Worm 5.625

Fly 25.587

16

Desrition of real networks?

Uetz et al. (2000) Nature 403, 623

Erdősi-Rényi Barabási-Albert

RandomRandom (RNN) (RNN) or scale-freeor scale-free (SFN) (SFN) network?network?

IDP functional advantages…IDP functional advantages…

……may also manifest in hub may also manifest in hub function?function?

- large binding surface

- adaptability (binding promiscuity)

- increased rate of interactions

- regulation by post-translational mod.

DIP 8

HUMANWORM YEASTFLY 10

What biological processes hubs are What biological processes hubs are involved in ?involved in ?

Further features in Further features in hubs ?hubs ?

HUMANWORM YEASTFLY

floating cutoff - top 10% -

(Yeast) hubs contain more of:

disorderrepeats

low-complexity length

Dosztányi (2006) J.Proteome. Res. 5, 2985

(Yeast) hubs contain a higher frequency of:

disorderrepeats

low-complexity

Dosztányi (2006) J.Proteome. Res. 5, 2985

Han et al. (2004) Nature 430, 88

„Party” and „date” hubs

Correlation coeff. of expression

party

date

Date hubs are more disordered than party hubs…

Ekman (2006) Genome Biol. 7, R45

Protein PONDR % STRING Partners

a -synuclein 100 27 parkin, tau, CaM

caldesmon 100 27 ERK, S100, myosin, actin, CAM

HMGA 100 18 AP1, NF-kB, C/EBPb , Oct-1, Sp1

synaptobrevin 100 8 syntaxin 1, BAP31, VAMP-ass. prot., SNAP-25

BRCA1 79 119 p53, ATM, BRCA2, c-Myc, Chk1

XPA 63 41 RPA70, RPA34, ERCC1, TFIIH, XAB1

estrogen receptor a 31 116 p53, BRCA1, CaM, c-Jun

p53 29 239 Mdm2, ATM, ERK, p38, BCL-Xl

Mdm2 26 72 p53, ARF, ATM, CK2, HIF-1a

calcineurin, subunit A 16 31 NFAT, calcipressin, cabin1, SOCS-3, calsarcin

14-3-3' x 12 97 p53, Wee1, tau, Raf-1, Cdc25c, Bad

Cdk2 7 125 PP2A, CycE1, DNA Pol a , BRCA1, cycA

actin 5 33 profilin, RNAse I, vit DBP, thymosin b4, cofilin

calmodulin 3 50 neurogranin, calcineurin, AC1, calponin, caldesmon

Dunker (2005) FEBS J. 272, 5129

Disorder inDisorder in hubhubss

HMGA (high mobility group A)

Reeves (2001) Gene 277, 63

IRF-1ATF/c-Jun

NF-YSRF

NF-kBp50

HIPK-2

Oct-6

PRGRP PRGRP PRGRP

HMGA: architectural transcription factor

HMGA

transcription

enhancosome cancer

overexpression

proliferation

differentiationchromatin remodeling

AT hook

Neg. gene reg.

competition

apoptosis

phosphorylation

growth factors

EGF, FGF, PDGF

environm. stress

hypoxia, virus

citokynes

IL-1b, IFN-tumor

promoters

phorbol ester

diff. factors

RA, Ca++ ionophores

Moonlighting: multiple functions of proteins

B

A

C

Ca++

Ca++ CST

AAA

BB

CC

The interplay of Ca2+ and CST in calpain regulation

Ca++

Ca++ CST

AAA

CC

The interplay of Ca2+ and CST in calpain regulation

CST subdomains A and C activate -calpain

- experiment: 2001 Sept. 11 -

0 10 20 30 40 50 60 70 80 900

10

20

30

40

50

60

A

C

peptide (M)

mu

-cal

pai

n a

ctiv

ity

(%)

A+C

Activation is a result of Ca2+-sensitisation

6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.20

20

40

60

80

100

mu-

calp

ain

activ

ity (%

)

pCa

A+C

6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.20

2

4

6

8

10

mu-

calp

ain

activ

atio

n ra

tiopCa

0’ 1’ 2’ 4’ 1’ 2’ 4’

MAP2c

Calpain activation in COS-7 cells

0

10

20

30

40

50

60

70

80

90

100

calp

ain

act

ivit

y

0 min PenA+PenC

20 M

Ca2+

1 mM

Ca2+1 mM

Ca2+

20 M

Ca2+

30 min PenA+PenC

Inhibition of Cdks in cell-cycle regulation

C y cA

C d k 2

- promoting assembly of complex

- activity within the complex

Excitation-contraction coupling in muscle

DHPR

Regulation of actin polymerisation

actin

thymosin b4 WH2

- promoting actin polymerisation

in actobindin (WH22)

and in ciboulot (WH23)

- activation of other target(s)

The securin The securin storystory

normal chromosome segregation

Inhibition of separase expression:

Waizenegger (2002) Curr. Biol. 12, 1368

The securin The securin storystory

Jallepalli (2001) Cell 105, 445

- securin knockout -

The securin story

ProteinOne (inhibitory)

function Another (activatory)

function Disorde

rIUPred (%)

PONDR® (%)

calpastatin inhibition of calpain activation of calpain * 100 80.1

CFTR

(R domain)inhibition of CFTR activation of CFTR * 40.8 53.7

DHPR

(peptide C)inhibition of RyR activation of of RyR * 100 81.1

EBV SMdown-regulation of intron-containing

mRNA

up-regulation of intron-less mRNA

44.1 44.4

MDM2

(180-298)down-regulation of

p21Cip1

activation of estrogen receptor a 70.0 70.0

p21Cip1/p27Kip1 inhibition of Cdk activation of Cdk * 48.2 61.0

PIAS1

(392-541)inhibition of activated

STATactivation of p53 41.3 40.7

PPI-2 inhibition of PP1 activation of PP1 * 91.7 77.8

ribosomal L5inhibition of MDM2

ubiquitin ligaseactivation/chaperoning of

ribosome * 22.3 30.8

securin inhibition of separaseactivation/chaperoning of

separase * 52.0 54.2

thymosin-b (WH2 domain)

sequestration of G-actin

activation of actin polymerization, ILK kinase * 100 64.3

Other multifarious Other multifarious effectorseffectors

TiBS 1996

The concept of moonlighting (multitasking)

The concept of moonlighting (multitasking)

PutA

PGI

neuroleukin

GAPDHUDGaconitase

IRE-BP

neuropilin

Cell type 1 Cell type 2

Jeffery (2004) DDT: Targets

F6P

G6P PGI

neuroleukin

Structural background of „classical” moonlighting

- the use of different surfaces of globular proteins -

Binding a partner in different conformations

Structural reorganisation around the partner

Binding different partners

Structural disorder throws new light on moonlighting

DHPR C

p21

WH2

Tompa et al. (2005) TiBS 30, 484

Fuzziness: disorder in the bound state

Curr. Opin. Struct. Biol. 2002

IDPs do fold upon binding

IDPs do fold upon binding …

CBP KIX

CREB KID

… but do they become fully folded?

CBP KIX

2400 AAsCREB KID

365 AAs

Kd: 3.1 M

… well, they don’t

Kd: 0.7 M

Tompa and Fuxreiter (2008) TiBS 33, 2-8

static dynamic

disorder

A B C D

Disorder in the bound state: fuzziness