Post on 04-Jan-2016
description
27041, Week 02Review of Week 01
27041, Introduction to Systems Biology2 CBS, Department of Systems Biology
The human genome sequencing project (HGP)
27041, Introduction to Systems Biology3 CBS, Department of Systems Biology
Systems Biology and emergent properties
27041, Introduction to Systems Biology4 CBS, Department of Systems Biology
Different model representations
Chen et al., Mol. Biol. Cell., 2004
27041, Introduction to Systems Biology6 CBS, Department of Systems Biology
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
27041, Introduction to Systems Biology7 CBS, Department of Systems Biology
Levels of organization
27041, Introduction to Systems Biology8 CBS, Department of Systems Biology
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.
Protein-protein interactions
27041, Introduction to Systems Biology10 CBS, Department of Systems Biology
Protein-protein interaction data is accumulating
27041, Introduction to Systems Biology11 CBS, Department of Systems Biology
30-40% Orphan Human Proteins
27041, Introduction to Systems Biology12 CBS, Department of Systems Biology
Protein-protein interactions: guilty-by-association
Protein-protein interaction network
Red protein: Unknown function
Yellow protein: RNA splicing
White protein: Other functional role
27041, Introduction to Systems Biology13 CBS, Department of Systems Biology
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.
27041, Introduction to Systems Biology14 CBS, Department of 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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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.
27041, Introduction to Systems Biology16 CBS, Department of Systems Biology
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 Biology17 CBS, Department of Systems BiologyCausier, 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 goes global
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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
27041, Introduction to Systems Biology21 CBS, Department of Systems Biology
841 Interactions
Ito et al. : ~ 6200 prey X ~ 6200 baits
Two large-scale Y2H studies: Ito et al.
Ito et al., PNAS 2001
27041, Introduction to Systems Biology22 CBS, Department of Systems Biology
841 Interactions
Ito et al. : ~ 6200 prey X ~ 6200 baits
692 Interactions
Uetz et al. : 6144 prey X 5345 baits
141551 700
Overlap
Reproducibility in Y2H
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Protein Complementation Assay (PCA)
27041, Introduction to Systems Biology24 CBS, Department of Systems Biology
Affinity Purification followed by Mass Spectrometry
(AP/MS)
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General strategy
Affi
nity
Purifi
catio
n 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
Affi
nity
Purifi
catio
n S
tep
Mass
Spect
rom
etr
y S
tep
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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
27041, Introduction to Systems Biology32 CBS, Department of Systems Biology
Mass spectrometry
Aebersold & Mann, Nature 2003
27041, Introduction to Systems Biology33 CBS, Department of Systems Biology
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
27041, Introduction to Systems Biology34 CBS, Department of Systems Biology
3617 interactions among 1578
proteins
Ho et al. : 725
3225 interactions among 1440
proteins
Gavin et al. : 1167 baits
1983007 3419
Overlap
1151052 (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
Scoring protein-protein 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
27041, Introduction to Systems Biology40 CBS, Department of 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)
27041, Introduction to Systems Biology41 CBS, Department of Systems Biology
Affinity Purification & mass spectrometryStrengths
• 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)
27041, Introduction to Systems Biology42 CBS, Department of Systems Biology
Filtering by subcellular localization
de Lichtenberg et al., Science, 2005