Transcript PowerPoint bemutat&#243

Protein networks,
moonlighting and fuzziness
Peter Tompa
Institute of Enzymology
Hungarian 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 method
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
Yeast two-hybrid method (Y2H)
bait
prey
Fields and Song (1989) Nature 340, 245
The yeast interactome
hub
node
edge
Uetz et al. (2000) Nature 403, 623
Comparison of TAP-tag and Y2H
Y2H
TAP-tag
- real complexes
- pairwise interactions
- tag interference
- no posttransl. mod.
- strength of interaction
- third partner?
- stoichiometry
- bait-prey nonprod.
- one condition
- compartmentalisation
- comparison?
- partial coverage!
How to relate Y2H and TAP-tag data?
TAP
tag
Y2H
B
A
B
A
E
C
L
F
G
H
I
J
C
A
A
x
E
K
A
x
H
“spoke” model better than “matrix”
TAP
tag
B
A
E
a-b, a-c, a-d, a-e
C
L
F
G
H
I
spoke (pairwise
interactions of bait):
J
K
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
Partial coverage (Y2H yeast data)
Deane et al. (2002) MCP 1, 349
Partial coverage (HTS vs. low-throughput)
Gandhi (2006) Nat. Gen. 38, 285
Partial coverage (all interactions)
Human 25.464
Yeast
16.069
Worm
5.625
Fly
16
25.587
Gandhi (2006) Nat. Gen. 38, 285
Desrition of real networks?
Erdősi-Rényi
Barabási-Albert
Uetz et al. (2000) Nature 403, 623
Random (RNN) or scale-free (SFN) network?
IDP functional advantages…
…may also manifest in hub function?
- large binding surface
- adaptability (binding promiscuity)
- increased rate of interactions
- regulation by post-translational mod.
DIP
8
HUMAN
WORM
YEAST
FLY
 10
What biological processes hubs are involved
in ?
Further features in hubs ?
HUMAN
WORM
YEAST
FLY
floating cutoff
- top 10% -
(Yeast) hubs contain more of:
disorder
repeats
low-complexity
length
Dosztányi (2006) J.Proteome. Res. 5, 2985
(Yeast) hubs contain a higher frequency of:
disorder
repeats
low-complexity
Dosztányi (2006) J.Proteome. Res. 5, 2985
„Party” and „date” hubs
date
party
Correlation coeff. of expression
Han et al. (2004) Nature 430, 88
Date hubs are more disordered than party hubs…
Ekman (2006) Genome Biol. 7, R45
Disorder in hubs
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 b 4, cofilin
calmodulin
3
50
neurogranin, calcineurin, AC1, calponin, caldesmon
Dunker (2005) FEBS J. 272, 5129
HMGA (high mobility group A)
PRGRP
PRGRP
PRGRP
IRF-1
ATF/c-Jun
SRF
NF-Y
NF-kB
p50
Oct-6
HIPK-2
Reeves (2001) Gene 277, 63
HMGA: architectural transcription factor
citokynes
IL-1b, IFN-g
phorbol
ester
environm. stress
hypoxia, virus
diff. factors
RA, Ca++
ionophores
growth factors
EGF, FGF, PDGF
tumor
promoters
HMGA
apoptosis
transcription
phosphorylat
ion
enhancosome
cancer
overexpression
Neg. gene reg.
competition
chromatin
remodeling
AT hook
proliferation
differentiation
Moonlighting: multiple functions of proteins
B
C
A
The interplay of Ca2+ and CST in calpain regulation
Ca++
Ca++
B
C
AA
CST
The interplay of Ca2+ and CST in calpain regulation
Ca++
Ca++
CST
C
AA
CST subdomains A and C activate -calpain
mu-calpain activity (%)
- experiment: 2001 Sept. 11 -
A+C
60
50
A
40
30
C
20
10
0
0
10
20
30
40
50
peptide (M)
60
70
80
90
100
80
A+C
0’
60
40
1’
MAP2c
20
0
6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2
pCa
2’
mu-calpain activation ratio
mu-calpain activity (%)
Activation is a result of Ca2+-sensitisation
4’
10
8
6
1’
2’
4’
4
2
0
6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2
pCa
Calpain activation in COS-7 cells
1 mM
1 mM
Ca2+
100
20 M
Ca2+
90
calpain activity
Ca2+
80
70
60
50
40
30
20
20 M
Ca2+
10
0
0 min
PenA+PenC
30 min
PenA+PenC
Inhibition of Cdks in cell-cycle regulation
Cdk2
- promoting assembly of complex
- activity within the complex
CycA
Excitation-contraction coupling in muscle
DHPR
Regulation of actin polymerisation
thymosin b4
WH2
actin
- promoting actin polymerisation
in actobindin (WH22)
and in ciboulot (WH23)
- activation of other target(s)
The securin story
normal chromosome segregation
Inhibition of separase expression:
Waizenegger (2002) Curr. Biol. 12, 1368
The securin story
- securin knockout -
Jallepalli (2001) Cell 105, 445
The securin story
Other multifarious effectors
Protein
One (inhibitory)
function
Another (activatory)
function
Disorde
r
IUPre
d (%)
PONDR
® (%)
calpastatin
inhibition of calpain
activation of calpain
100
80.1
inhibition of CFTR
activation of CFTR
*
*
40.8
53.7
inhibition of RyR
activation of of RyR
*
100
81.1
EBV SM
down-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
STAT
activation of p53
41.3
40.7
PPI-2
inhibition of PP1
activation of PP1
91.7
77.8
22.3
30.8
52.0
54.2
100
64.3
CFTR
(R domain)
DHPR
(peptide C)
activation/chaperoning of
ribosome
activation/chaperoning of
securin
inhibition of separase
separase
activation of actin
thymosin-b (WH2 sequestration of Gdomain)
actin
polymerization, ILK kinase
ribosomal L5
inhibition of MDM2
ubiquitin ligase
*
*
*
*
*
TiBS 1996
The concept of moonlighting (multitasking)
The concept of moonlighting (multitasking)
PutA
neuroleukin
PGI
neuropilin
aconitase
IRE-BP
GAPDH
UDG
Structural background of „classical”
moonlighting
- the use of different surfaces of globular proteins -
neuroleukin
F6P
G6P
Cell type 1
PGI
Cell type 2
Jeffery (2004) DDT: Targets
Structural disorder throws new light on moonlighting
Tompa et al. (2005) TiBS 30, 484
Binding a partner in different conformations
DHPR C
Binding different partners
WH2
Structural reorganisation around the partner
p21
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 AAs
CREB KID
365 AAs
… well, they don’t
Kd: 3.1 M
Kd: 0.7 M
Disorder in the bound state: fuzziness
static
A
dynamic
B
C
D
disorder
Tompa and Fuxreiter (2008) TiBS 33, 2-8