27041, Week 02. Review of Week 01

Transcription

1 27041, Week 02 Review of Week 01

2 The human genome sequencing project (HGP) 2 CBS, Department of Systems Biology

3 Systems Biology and emergent properties 3 CBS, Department of Systems Biology

4 Different model representations 4 CBS, Department of Systems Biology Chen et al., Mol. Biol. Cell., 2004

5 How radios work and how to fix them... Lazebnik, Cancer Cell, CBS, Department of Systems Biology

6 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 Sequencing Gene knock-out Microarrays Protein-Protein interactions Protein-DNA interactions Subcellular Localization Microarrays Proteomics Metabolomics Model Generation 6 CBS, Department of Systems Biology

7 Levels of organization 7 CBS, Department of Systems Biology

8 Networks in Molecular Biology Protein-Protein interactions Protein-DNA interactions Genetic interactions Metabolic reactions Text mining interactions Association Networks Etc. Barabasi & Oltvai, Nature Reviews, CBS, Department of Systems Biology

9 Protein-protein interactions

10 Protein-protein interaction data is accumulating 10 CBS, Department of Systems Biology

11 30-40% Orphan Human Proteins 11 CBS, Department of Systems Biology

12 Protein-protein interactions: guilty-byassociation Protein-protein interaction network Red protein: Unknown function Yellow protein: RNA splicing White protein: Other functional role 12 CBS, Department of Systems Biology

13 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. 13 CBS, Department of Systems Biology

14 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 14 CBS, Department of Systems Biology

15 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. 15 CBS, Department of Systems Biology

16 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) 16 CBS, Department of Systems Biology

17 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, CBS, Department of Systems Biology

18 Y2H goes global 18 CBS, Department of Systems Biology

19 Y2H Random Library a Approach Bait B1 X Genomic fragment library Interacting Domain Protein Selected fragments (prey) 19 CBS, Department of Systems Biology

20 Two large-scale Y2H studies: Uetz et al. Uetz et al. : 6144 prey X 5345 baits 692 Interactions Uetz et al, Nature CBS, Department of Systems Biology

21 Two large-scale Y2H studies: Ito et al. Ito et al. : ~ 6200 prey X ~ 6200 baits 841 Interactions Ito et al., PNAS CBS, Department of Systems Biology

22 Reproducibility in Y2H Uetz et al. : 6144 prey X 5345 baits Ito et al. : ~ 6200 prey X ~ 6200 baits 692 Interactions 841 Interactions Overlap 22 CBS, Department of Systems Biology

23 Protein Complementation Assay (PCA) 23 CBS, Department of Systems Biology

24 Affinity Purification followed by Mass Spectrometry (AP/MS) 24 CBS, Department of Systems Biology

25 General strategy Affinity Purification Step 25 CBS, Department of Systems Biology

26 Affinity Purification 26 CBS, Department of Systems Biology

27 Affinity Chromatography Designed to purify a protein from a complex mixture Load affinity column with antigen (or antibody) Proteins sieve through matrix of affinity beads Proteins react with different affinities 27 CBS, Department of Systems Biology

28 Affinity Chromatography (2) Wash off proteins that do not bind Elute and collect bound proteins 28 CBS, Department of Systems Biology

29 General strategy Mass Spectrometry Step Affinity Purification Step 29 CBS, Department of Systems Biology

30 Mass spectrometry Aebersold & Mann, Nature, CBS, Department of Systems Biology

31 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, CBS, Department of Systems Biology

32 Mass spectrometry Aebersold & Mann, Nature CBS, Department of Systems Biology

33 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 33 CBS, Department of Systems Biology

34 Reproducibility in AP/MS Gavin et al. : 1167 baits Ho et al. : interactions among 1440 proteins 3617 interactions among 1578 proteins Overlap in baits 1052 (454) (493) Overlap 34 CBS, Department of Systems Biology

35 35 CBS, Department of Systems Biology

36 Recent HTP (binary) PPI networks Y2H by Yu et al : 2018 proteins, 2930 interactions PCA by Tarassov et al : 1124 proteins, 2770 interactions 36 CBS, Department of Systems Biology

37 Scoring protein-protein interactions

38 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 CBS, Department of Systems Biology

39 Benchmarking interaction-scores 39 CBS, Department of Systems Biology

40 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) 40 CBS, Department of Systems Biology

41 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) 41 CBS, Department of Systems Biology

42 Filtering by subcellular localization de Lichtenberg et al., Science, CBS, Department of Systems Biology