27041, Week 02 - CBS · 28 CBS, Department of Systems Biology 27041, Introduction to Systems...
Transcript of 27041, Week 02 - CBS · 28 CBS, Department of Systems Biology 27041, Introduction to Systems...
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The human genome sequencing project (HGP)
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Systems Biology and emergent properties
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Different model representations
Chen et al., Mol. Biol. Cell., 2004
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How radios work and how to fix them...
Lazebnik, Cancer Cell, 2002
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Model
Generation
Systems Biology at a glance
Parts List
YER001W YBR088C
YOL007C
YPL127C
YNR009W
YDR224C
YDL003W
YBL003C
…
YDR097C YBR089W
YBR054W
YMR215W
YBR071W
YBL002W
YNL283C
YGR152C
…
• Sequencing
• Gene knock-out
• Microarrays
Interactions
• Protein-Protein interactions
• Protein-DNA interactions
• Subcellular Localization
Dynamics
• Microarrays
• Proteomics
• Metabolomics
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Networks in Molecular Biology
Barabasi & Oltvai, Nature Reviews, 2004
• Protein-Protein interactions
• Protein-DNA interactions
• Genetic interactions
• Metabolic reactions
• Text mining interactions
• Association Networks
• Etc.
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Protein-protein interaction data is accumulating
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30-40% Orphan Human Proteins
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Protein-protein interactions: guilty-by-association
Protein-protein interaction network
Red protein: Unknown function
Yellow protein: RNA splicing
White protein: Other functional role
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Classical methods for identifying protein-protein 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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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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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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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)
Transcription factor
27041, Introduction to Systems Biology 17 CBS, Department of Systems Biology Causier, Mass spectrometry Reviews, 2004
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.
Yeast Two-Hybrid Method
Animation!
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Y2H Random Library a Approach
Bait B1 X Genomic fragment library
Protein
Selected fragments (prey)
Interacting Domain
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692 Interactions
Uetz et al. : 6144 prey X 5345 baits
Two large-scale Y2H studies: Uetz et al.
Uetz et al, Nature 2000
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841 Interactions
Ito et al. : ~ 6200 prey X ~ 6200 baits
Two large-scale Y2H studies: Ito et al.
Ito et al., PNAS 2001
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841 Interactions
Ito et al. : ~ 6200 prey X ~ 6200 baits
692 Interactions
Uetz et al. : 6144 prey X 5345 baits
141 551 700
Overlap
Reproducibility in Y2H
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Protein Complementation Assay (PCA)
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Affinity Purification followed by Mass Spectrometry
(AP/MS)
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General strategy
Affinity Purification S
tep
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Affinity Chromatography
Load affinity column with antigen (or antibody)
Designed to purify a protein from a complex mixture
Proteins sieve through matrix of affinity beads
Proteins react with different affinities
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Affinity Chromatography (2)
Wash off proteins that do not bind Elute and collect bound proteins
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General strategy
Affinity Purification S
tep M
ass
Spe
ctro
met
ry S
tep
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Mass spectrometry
Aebersold & Mann,
Nature, 2003
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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
For details on Proteomics, see Aebersold & Mann, Nature, 2003
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Mass spectrometry
Aebersold & Mann, Nature 2003
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Two large-scale mass spec experiments
Gavin et al. Ho et al.
589 protein complexes
(232 distinct)
Gavin et al. : 1167 baits
3617 interactions among 1578
proteins
Ho et al. : 725 baits
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3617 interactions among 1578
proteins
Ho et al. : 725
3225 interactions among 1440
proteins
Gavin et al. : 1167 baits
198 3007 3419
Overlap
115 1052 (454)
610 (493)
Overlap in baits
Reproducibility in AP/MS
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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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Topology based scoring of interactions
Low confidence (4 unshared interaction partners)
High confidence (1 unshared interaction partners)
A B C
Yeast two-hybrid
Low confidence (rarely purified together)
High confidence (often purified together)
Complex pull-downs
D
de Lichtenberg et al., Science 2005
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Benchmarking interaction-scores
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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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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)