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Interactome Under Construction
Interactome Under Construction
Interactome under
tag, along with any that interact with them.
Interactions between the RL-fused bait and
the Flag-tagged prey are detected when light
is emitted. Other binary methods include the
construction
mammalian protein–protein interaction trap
and techniques based on proteome chips.
The most common co-complex method is
co-immunoprecipitation (coIP) coupled with
mass spectrometry (MS). In this approach, a
protein bait is tagged with a molecular marker.
Developing techniques are helping researchers to build the Several types of tags are commercially available;
protein interaction networks that underlie all cell functions. each requires a distinct biochemical technique
to recognize the tag and fish the bait protein out
of the cell lysate, bringing with it any interacting
b y L a u r a b o n e t ta open-source software for network visualiza- proteins. These are then identified by MS.
A
tion. “That effort identified 30,000 genes, but In addition to these empirical methods,
n old adage says: “Show me your friends, that is not the end goal. How the genes work in researchers have used computational techniques
and I’ll know who you are.” In the same pathways and how these pathways function in to predict interactions on the basis of factors
way, finding interaction partners for disease states and development is the end goal. such as amino-acid sequence and structural
a protein can reveal its function. To that end, To accomplish this we will need to systemati- information. “People ask ‘Why are you predict-
researchers are now building entire networks of cally map gene and protein interactions.” ing interactions when you can just do the exper-
protein–protein interactions. Unlike biological Unlike the genome, the interactome — the iment?’” says Gary Bader, a bioinformatician at
pathways, which represent a sequence of molec- set of protein-to-protein interactions that the University of Toronto. “But experimental
ular interactions leading to a final result — for occurs in a cell — is dynamic. Many inter- techniques fail for some proteins.”
example, a signalling cascade — networks are actions are transient, and others occur only
interlinked. Represented as starbursts of pro- in certain cellular contexts or at particular False Readings
tein ‘nodes’ linked by interaction ‘edges’ to form times in development. The interactome may Every step of a procedure to detect protein–
intricate constellations, they provide insight into be tougher to solve than the genome, but the protein interactions — from the reagents used
the mechanisms of cell functions. Furthermore, information, researchers say, is crucial for a to the cell types and experimental conditions —
placing proteins encoded by disease genes into complete understanding of biology. influences the proteins that are identified. Two
these networks will let researchers determine studies this year used similar methods to iden-
the best candidates for assessing disease risk The RighT PaRTneRs tify interacting proteins in transcription factors
and targeting with therapies. At any time, a human cell may contain about in embryonic stem cells3,4; there was incomplete
“This is the next step after the Human 130,000 binary interactions between proteins1. overlap between the resulting data sets. “If you
Genome Project,” says Trey Ideker, a systems So far, a mere 33,943 unique human protein– use the same protocol you will get reproducible
biologist at the University of California, San protein interactions are listed on BioGRID lists of proteins. But different labs use different
Diego, and principal investigator at the National (http://thebiogrid.org), a database that stores protocols, which affects the end result,” says
Resource for Network Biology, which provides interaction data. Clearly, there is work to do. Raymond Poot, a cell biologist at Erasmus MC
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© 2010 Macmillan Publishers Limited. All rights reserved
technology Protein–Protein interactions
hospital in Rotterdam, the Netherlands, and Calling out false positives — reported inter- detect the interactions. But the definition of a
lead author of one of the studies. actions that don’t actually occur — and false ‘real’ interaction depends on the context. “Does
In his protocol, Poot pulled interacting pro- negatives — interactions that do occur but are a real interaction mean that two proteins inter-
teins from cells using nuclear extracts express- not picked up by the experimental protocol or act if they are placed next to each other in a
ing different Flag-tagged transcription factors. are discarded — is one of the main challenges test tube, or that they must interact in a cell? Or
He added a nuclease to his reactions to remove in the field. “Normally when you do a coIP fol- does real mean that the interaction should have
DNA and eliminate possible artefacts caused lowed by MS you will get hundreds of protein a biological function?” asks Ideker. Researchers
by proteins binding to it. “Transcription fac- candidates interacting with any one bait,” says can home in on functional interactions by com-
tors bind to DNA so you are likely to pull out Wade Harper, a cell biologist at Harvard Medi- bining data on interactions with other types of
DNA-binding factors that are not directly inter- cal School in Boston, Massachusetts. “When biological information, such as genetic interac-
acting,” he explains. Purifying many different you weed out all the stochastic and non-specific tions, protein localizations or gene expression.
transcription factors with the same protocol interactions you end up with many fewer pro- For instance, proteins whose genes are co-
also enabled the researchers to determine which teins. Some proteins in large complexes might expressed are likely to interact with each other
interactions were most likely to be specific. For have 30–50 partners, others only 4–5.” or to be part of the same complex or pathway.
example, proteins that consistently co-purified One way in which researchers increase the Many tools are available on the web for inte-
with all transcription factors would be treated accuracy of their results is to use more than one grating different types of information about a
as unlikely to indicate a genuine interaction. method (for example, Y2H plus LUMIER) to given protein or gene. One is GeneMANIA,
developed by Bader’s group in collaboration
with Quaid Morris, a computational biolo-
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© 2010 Macmillan Publishers Limited. All rights reserved
Protein–Protein interactions technology
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© 2010 Macmillan Publishers Limited. All rights reserved
technology Protein–Protein interactions
real-time analysis
paCiFiC BiOSCienCeS
in november, pacific Biosciences of Menlo technology can detect relatively
park, California, commercially released weak interactions,” says Jonas
its third-generation Dna-sequencing Korlach, a scientific fellow at
platform, based on its single-molecule, pacific Biosciences, adding that
real-time (SMrt) technology. a single Dna it could pick out interactions
polymerase bound to a Dna template is that happen so quickly that they
attached to a tiny chamber illuminated can’t be identified by current
by lasers, and nucleotides labelled with methods.
coloured fluorophores are introduced to as a step towards such applications,
it. as the polymerase incorporates them, Joseph puglisi, a structural biologist at
each base is held for a few microseconds, Stanford University School of Medicine in
while the fluorophore emits coloured light California, and his group, with scientists at Future SMRT systems could reveal interactions.
corresponding to the base identity. SMrt pacific Biosciences, observed transfer rnas
technology could also be used to analyse binding to single ribosomes in real time13. in protein factors to determine how the
biomolecules other than Dna, and could an unpublished follow-up, puglisi’s group has translation machinery synthesizes proteins.
become a common tool for detecting protein used SMrt technology to watch interactions “We have just seen the tip of the iceberg in
interactions, with some unique features. “this between transfer rnas, ribosomes and terms of applications,” says Korlach. L.b.
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© 2010 Macmillan Publishers Limited. All rights reserved