27041, Week 02
Review of Week 01
The human genome sequencing project
(HGP)
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Systems Biology and emergent properties
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Different model representations
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Chen et al., Mol. Biol. Cell., 2004
27041, Introduction to Systems Biology
How radios work and how to fix them...
Lazebnik, Cancer Cell, 2002
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Systems Biology at a glance
YER001W
YDR097C
YBR088C
YBR089W
YOL007C
YBR054W
YPL127C
YMR215W
YNR009W
YBR071W
YDR224C
YBL002W
YDL003W
YNL283C
YBL003C
YGR152C
…
…
Parts List
•  Sequencing
•  Gene knock-out
•  Microarrays
•  Protein-Protein interactions
•  Protein-DNA interactions
Interactions
•  Subcellular Localization
Model
Generation
•  Microarrays
•  Proteomics
•  Metabolomics
Dynamics
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27041, Introduction to Systems Biology
Levels of organization
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Networks in Molecular Biology
•  Protein-Protein interactions
•  Protein-DNA interactions
•  Genetic interactions
•  Metabolic reactions
•  Text mining interactions
•  Association Networks
•  Etc.
Barabasi & Oltvai, Nature Reviews, 2004
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Protein-protein interactions
Protein-protein interaction data is
accumulating
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
30-40% Orphan Human Proteins
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Protein-protein interactions: guilty-byassociation
Protein-protein interaction network
Red protein:
Unknown function
Yellow protein: RNA splicing
White protein: Other functional role
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Classical methods for identifying proteinprotein interactions
•  Co-immunoprecipitation
•  Affinity chromatography / crosslinking
•  Fluorescence energy transfer (FRET)
•  Dominant negatives
–  Over-expression of a mutant form of protein X causes loss of function
despite the presence of native proteins. One explanation is that X
forms a multimer that sequesters functional proteins.
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
High-throughput methods for measuring
interactions
•  Phage display
•  SOS recruitment assay
•  Split-ubiquitin system
•  Dual-bait system
•  2-hybrid
•  Protein complementation assay (PCA)
•  Co-immunoprecipitation
•  Protein arrays
•  ChIP-Chip/Chip-Seq
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Yeast Two Hybrid (Y2H) Method
•  One problem with phage display and other in vitro technologies is that
the measured binding may not actually occur.
•  Y2H assays interactions in vivo.
•  Uses property that transcription factors generally have separable
transcriptional activation (AD) and DNA binding (DBD) domains.
•  A functional transcription factor can be created if a separately expressed
AD can be made to interact with a DBD.
•  A protein ‘bait’ B is fused to a DBD and screened against a library of
protein ‘preys’, each fused to a AD.
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Transcription factor
An activating
transcription factor:
1.  Binds to DNA using a
DNA-binding domain
(DBD)
2.  Recruits the
transcriptional
machinery using a
transcriptional
activation domain
(AD)
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Yeast Two-Hybrid Method
Y2H assays interactions in vivo.
Uses property that transcription
factors generally have separable
transcriptional activation (AD) and
DNA binding (DBD) domains.
A functional transcription factor
can be created if a separately
expressed AD can be made to
interact with a DBD.
A protein ‘bait’ B is fused to a DBD
and screened against a library of
protein ‘preys’, each fused to a AD.
Animation!
Causier, Mass spectrometry Reviews, 2004
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27041, Introduction to Systems Biology
Y2H goes global
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Y2H Random Library a Approach
Bait B1
X
Genomic fragment library
Interacting Domain
Protein
Selected
fragments
(prey)
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Two large-scale Y2H studies: Uetz et al.
Uetz et al. : 6144 prey X
5345 baits
692
Interactions
Uetz et al, Nature 2000
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Two large-scale Y2H studies: Ito et al.
Ito et al. : ~ 6200 prey X ~
6200 baits
841
Interactions
Ito et al., PNAS 2001
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Reproducibility in Y2H
Uetz et al. : 6144 prey X
5345 baits
Ito et al. : ~ 6200 prey X ~
6200 baits
692
Interactions
841
Interactions
551
141
700
Overlap
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27041, Introduction to Systems Biology
Protein Complementation Assay (PCA)
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Affinity Purification followed
by Mass Spectrometry
(AP/MS)
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27041, Introduction to Systems Biology
General strategy
Affinity Purification Step
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Affinity Purification
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Affinity Chromatography
Designed to purify a protein from a complex mixture
Load affinity column
with antigen
(or antibody)
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Proteins react with
different affinities
Proteins sieve
through matrix of
affinity beads
27041, Introduction to Systems Biology
Affinity Chromatography (2)
Wash off proteins that
do not bind
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Elute and collect bound proteins
27041, Introduction to Systems Biology
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Affinity Purification Step
Mass Spectrometry Step
General strategy
27041, Introduction to Systems Biology
Mass spectrometry
Aebersold & Mann,
Nature, 2003
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Mass spectrometry
•  Mass spectrometers consist of three essential parts:
–  Ionization source: Converts peptides into gas-phase ions (MALDI +
ESI)
–  Mass analyzer: Separates ions by mass to charge (m/z) ratio (Ion
trap, time of flight, quadrupole)
–  Ion detector: Current over time indicates amount of signal at each m/
z value
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For details on Proteomics, see Aebersold & Mann, 27041,
Nature,
2003
Introduction to Systems Biology
CBS, Department of Systems Biology
Mass spectrometry
Aebersold & Mann, Nature 2003
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Two large-scale mass spec experiments
Gavin et al.
Ho et al.
Gavin et al. : 1167 baits
589 protein
complexes
(232 distinct)
Ho et al. : 725 baits
3617
interactions
among 1578
proteins
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Reproducibility in AP/MS
Gavin et al. :
1167 baits
Ho et al. :
725
3225
interactions
among 1440
proteins
3617
interactions
among 1578
proteins
Overlap in baits
1052
(454)
115
610
(493)
3007
198
3419
Overlap
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Recent HTP (binary) PPI networks
Y2H by Yu et al. 2008 : 2018 proteins, 2930 interactions
PCA by Tarassov et al. 2008 : 1124 proteins, 2770 interactions
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Scoring protein-protein interactions
Topology based scoring of interactions
Yeast twohybrid
D
A
B
C
High confidence (1 unshared interaction partners)
Low confidence (4 unshared interaction partners)
Complex pull-downs
Low confidence (rarely purified together)
High confidence (often purified together)
de Lichtenberg et al., Science 2005
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Benchmarking interaction-scores
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Issues with Y2H
•  Strengths
–  high sensitivity (transient & permanent PPIs)
–  takes place in vivo
–  independent of endogenous expression
•  Weaknesses: False positive interactions
–  Auto-activation
–  ‘sticky’ prey
–  detects “possible interactions” that may not take place under real
physiological conditions
–  may identify indirect interactions (A-C-B)
•  Weaknesses: False negatives interactions
–  Similar studies often reveal very different sets of interacting proteins
(i.e. False negatives)
–  may miss PPIs that require other factors (e.g. ligands, proteins,
PTMs)
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Affinity Purification & mass spectrometry
Strengths
•  high specificity
•  well suited for detecting permanent or
strong transient interactions (complexes)
•  detects real, physiologically relevant PPIs
Weaknesses
•  less suited for detecting weaker transient
interactions (low sensitivity)
•  may miss complexes not present under the
given experimental conditions (low
sensitivity)
•  may identify indirect interactions (A-C-B)
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology
Filtering by subcellular localization
de Lichtenberg et al., Science, 2005
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CBS, Department of Systems Biology
27041, Introduction to Systems Biology