_________ ____
Interactions and more interactions
Rob Russell
Cell Networks
University of Heidelberg
Russell Group, Protein Evolution
_________ ____
Russell Group, Protein Evolution
Aloy & Russell Nature Rev Mol Cell Biol 2006
_________ ____
But instead of a cell dominated by randomly
colliding individual protein molecules, we now
know that nearly every major process in a cell
is carried out by assemblies of 10 or more
protein molecules
Bruce Alberts, Cell 1998
Russell Group, Protein Evolution
____
Yeast two-hybrid _________
system
Fields & Song, Nature, 340, 245, 1989
a
Native GAL4
UASG
GAL1-lacZ
b
c
Individual hybrids with GAL4 domains
Interaction between hybrids reconstitutes GAL4 activity
GAL4 DNAbinding domain
X
UASG
GAL1-lacZ
GAL4 DNAbinding domain
X
UASG
Y
GAL1-lacZ
GAL4 activating region
Y
UASG
GAL1-lacZ
Russell Group, Protein Evolution
Applied to whole Yeast genome
Uetz et al, Nature, 403, 623, 2000.
Ito et al, PNAS, 98, 4569, 2001.
_________
Interaction discovery
I
The two-hybrid system
Binary interactions:
Bait
Prey
FUS3
DIG2
DIG2
FUS3
x1000s LSM2
PAT1
CKS1
CLB1
NPL4
UFD1
NPL4
CDC48
NPL4
FUC1
NPL4
SUA7
.
.
.
Russell Group, Protein Evolution
.
.
.
Uetz et al, Nature, 2000. (Yeast)
Ito et al, PNAS, 2001. (Yeast)
Rain et al, Nature, 2002. (H.pylori)
Giot et al, Science, 2003 (D. melanogaster)
Li et al, Science, 2004 (C. elegans)
____
_________
The system works,
but how?
____
c
Interaction between hybrids reconstitutes GAL4 activity
GAL4 DNAbinding domain
Y
X
UASG
Gal-4 (C)
(hypothetical)
GAL1-lacZ
Native GAL4
CDC28
Cyclin A
S12
CKS
Gal-4 (N)
Russell Group, Protein Evolution
L22
_________ ____
Two datasets in Yeast
See:
Ito et al, PNAS, 2001
(comparing to Uetz et al, Nature, 2000)
Russell Group, Protein Evolution
_________
____
Interaction discovery
II
Affinity purification (e.g. TAP/MS)
x1000s
l
100
Relative Intensity [%]
Complexes:
Bait
Co-purification partners
FUS3
DIG2
DIG1
DIG3
DIG2
FUS3
DIG2
NPL4
UFD1
CDC48 FUC1…
(Etc.)
50
l
l
l
l
l
l M
M
l
1000
l
l
*
l
l
l
l
1500
2000
*
l
l
2500
3000
m/z
Russell Group, Protein Evolution
Gavin et al, Nature, 2002. (Yeast)
Ho et al, Nature, 180, 2002. (Yeast)
Trying to define binary
_________
interactions from purification
data
____
Reality
Purification
Spoke
Matrix
Purifications only report a collection
of proteins and don’t provide any
information about precisely who
interacts with whom.
There are thus two models for
representing binary interactions
from complexes, neither of which
are real.
Hakes et al, Comp Funct Genomics, 2006
Russell Group, Protein Evolution
y
Different worlds
Total
_________ ____
Intersection with 3D ( Transient Total Complexes )
3D structure
Two-hybrids
Affinity purification
Homology
Aloy
Ito
Uetz
Ho
Gavin
8597
420 499 79
781
23 25 2
69 130 61
12 138 126
499
499
011
561
10 27 17
6 23 17
4475
8
1
1
231
330
Comparing interactions to known 3D structures
shows that original
yeast 0 1
1447
25
6 contain more
199transient interactions,
9 10 1
352
two-hybrid
datasets
compared
to affinity
72690
130
27 that contain
106
92
3 31 28
purification
datasets
more stable complexes
138of 25 Uetz et23
113
97 23 are transient,
4197 2 are 48751
(e.g.
al interactions
with structures,
dedicated or stable)
Intersection with each other
Russell Group, Protein Evolution
Total
Aloy & Russell, Trends Biochem Sci, 2003
_________ ____
Error rates in interaction discovery
False negatives: interactions known to occur that are missed by
a screen
- To asses this one needs a reference set of positives (i.e.
known interactions) among a set of proteins being screened.
The fraction of these missed is the false-negative rate. Relatively
simple
- normally one has a set of previously determined interactions or
“gold standard”
False positives: interactions reported by a screen that are
incorrect
- To assess this one needs a set of interactions that are known
not to occur that are seen in a screen. Very difficult to obtain –
how can you know that two proteins definitely do not interact?
- tricks include taking pairs of proteins presumed to never see
each other (i.e. different cellular compartment, etc.)
Russell Group, Protein Evolution
Von Mering et al, Nature, 2002
_________
____
Error rates in interaction
discovery:
the old view
Russell Group, Protein Evolution
Von Mering et al, Nature, 2002
_________
____
Error rates in interaction
discovery:
the new view
Russell Group, Protein Evolution
Yu et al, Nature, 2002
Sociological bias _________
affects the
perceived performance
____
Interactions determined on a protein
by protein basis are focused around
what the investigator wants to study,
and thus biased towards particular
areas of biology that are hot.
High-throughput techniques are used
precisely to find new interactions.
Thus
using
the
previously
determined networks as a “gold
standard” is likely to be unfair.
Russell Group, Protein Evolution
Braun et al, Nature Methods, 2008
_________ ____
Interaction data: predictions I
Proteins in the same bacterial operon
are typically functionally associated,
and often physically interacting.
Groups of proteins entirely absent in
one or more organisms among a
closely related set
are often
functionally/physically associated
Russell Group, Protein Evolution
Proteins that are separate in some
organisms and fused in others are likely
interacting physically.
Aloy & Russell, Nature Rev Mol Cell Biol 2006
_________ ____
Interaction data: predictions II
Pairs of proteins homologous to pairs of
proteins seen to interact in known 3D
structures can interact in the same way.
Pairs of proteins containing a pair of
domains often seen in interacting
proteins can be used to infer
interactions
in
proteins
where
interactions have not been observed.
Russell Group, Protein Evolution
The presence of a linear motif can
indicate interactions with proteins
known to bind this motif..
Aloy & Russell, Nature Rev Mol Cell Biol 2006
_________
____
Interaction databases
Resources are very different in appearance and content
Efforts are underway to make a unified search/view, but not complete
Thus one needs currently to look at several sites to check if an interaction is
known
Some are content (e.g. IntAct, MINT) others are processed and augmented
(e.g. STRING) with additional predicted/inferred interactions
Russell Group, Protein Evolution
_________
Interaction networks
____
Grb-2
Sos-1
RGS-4
Ga/q
Node
Edge
RGS-3
Russell Group, Protein Evolution
Node
_________ ____
Biological interaction networks
Node
Node
Edge
Nodes:
•Proteins
•Genes
•Chemicals
•Effects(?)
Edges:
•Physical interaction (e.g. yeast two-hybrid)
•Co-expression (e.g. microarrays)
•Same operon
•Regulation of gene expression (protein to gene)
•Catalysis (e.g. metabolic networks)
Russell Group, Protein Evolution
_________
____
Interaction networks
Jeong et al,
Nature, 2001.
Biological networks tend to be scale free: most nodes (e.g.
proteins) are connected to only a few others with a handful of
“hubs” making many more interactions.
They are also “small-world” in that any pair of nodes tends to be
connected via a relatively small number of intermediate nodes.
Russell Group, Protein Evolution
_________
____
“Hubs” in networks
Hubs are more likely to be lethal when deleted
Jeong et al, Nature, 2001
Hubs are more likely to be disordered.
Haynes et al, PLoS Comp Biol, 2006
Russell Group, Protein Evolution
_________ ____
p53 – the promiscuous transcription factor
Russell Group, Protein Evolution
_________
____
Linear motifs in p53
P 15:DNA-PK,RSK2,ATM
P 18:CK1s
NES
P 20:CHK2
9:Unknown
P
MDM2
P 55:MAPKs
37:DNA-PK/ATM P
CYCLIN
S 386
DNA binding domain (95-289)
33:GSK-3s,CDK7,CDKs
P
USP7
Tetramerization
domain (323-356)
P 46:HIPK2
IUPred disorder prediction
Russell Group, Protein Evolution
P
215:AuroraA
P
P
315:AuroraA,CDKs
P
P
371,376,378:CDK7
P
392:CDK2s,CDK7,EIF2AK2
Russell & Gibson, FEBS Lett. 2008