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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CBS, Department of Systems Biology
Chen et al., Mol. Biol. Cell., 2004
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
…
…
Interactions
Dynamics
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CBS, Department of Systems Biology
•
Sequencing
•
Gene knock-out
•
Microarrays
•
Protein-Protein interactions
•
Protein-DNA interactions
•
Subcellular Localization
•
Microarrays
•
Proteomics
•
Metabolomics
Model
Generation
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
•
•
•
•
•
•
•
•
•
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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
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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CBS, Department of Systems Biology
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)
19
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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CBS, Department of Systems Biology
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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CBS, Department of Systems Biology
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 Chromatography
Designed to purify a protein from a complex mixture
Load affinity column
with antigen
(or antibody)
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CBS, Department of Systems Biology
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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CBS, Department of Systems Biology
Elute and collect bound proteins
27041, Introduction to Systems Biology
29
CBS, Department of Systems Biology
Affinity Purification Step
Mass Spectrometry Step
General strategy
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
31
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
34
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
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