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