1 / 37

27041, Week 02

27041, Week 02. Review of Week 01. The human genome sequencing project (HGP). Systems Biology and emergent properties. Different model representations. Chen et al., Mol. Biol. Cell., 2004. Model Generation. Systems Biology at a glance. YER001W YBR088C YOL007C YPL127C YNR009W YDR224C

hidalgo-orrego
Download Presentation

27041, Week 02

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 27041, Week 02 Review of Week 01

  2. The human genome sequencing project (HGP)

  3. Systems Biology and emergent properties

  4. Different model representations Chen et al., Mol. Biol. Cell., 2004

  5. Model Generation Systems Biology at a glance YER001W YBR088C YOL007C YPL127C YNR009W YDR224C YDL003W YBL003C … YDR097C YBR089W YBR054W YMR215W YBR071W YBL002W YNL283C YGR152C … • Sequencing • Gene knock-out • Microarrays Parts List • Protein-Protein interactions • Protein-DNA interactions • Subcellular Localization Interactions • Microarrays • Proteomics • Metabolomics Dynamics

  6. Levels of organization

  7. 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

  8. Protein-protein interactions

  9. Protein-protein interaction data is accumulating

  10. 30-40% Orphan Human Proteins

  11. Protein-protein interactions: guilty-by-association Protein-protein interaction network Red protein: Unknown function Yellow protein: RNA splicing White protein: Other functional role

  12. 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.

  13. 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. 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. Transcription factor An activating transcription factor: Binds to DNA using a DNA-binding domain (DBD) Recruits the transcriptional machinery using a transcriptional activation domain (AD)

  16. 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

  17. Y2H goes global

  18. Y2H Random Library a Approach X Genomic fragment library Bait B1 Protein Interacting Domain Selected fragments (prey)

  19. Uetz et al. : 6144 prey X 5345 baits 692 Interactions Two large-scale Y2H studies: Uetz et al. Uetz et al, Nature 2000

  20. Ito et al. : ~ 6200 prey X ~ 6200 baits 841 Interactions Two large-scale Y2H studies: Ito et al. Ito et al., PNAS 2001

  21. 551 700 141 Overlap Reproducibility in Y2H Uetz et al. : 6144 prey X 5345 baits Ito et al. : ~ 6200 prey X ~ 6200 baits 692 Interactions 841 Interactions

  22. Protein Complementation Assay (PCA)

  23. Affinity Purification followed by Mass Spectrometry (AP/MS)

  24. General strategy Affinity Purification Step

  25. Load affinity column with antigen (or antibody) Proteins react with different affinities Proteins sieve through matrix of affinity beads Affinity Chromatography Designed to purify a protein from a complex mixture

  26. Wash off proteins that do not bind Elute and collect bound proteins Affinity Chromatography (2)

  27. General strategy Affinity Purification Step Mass Spectrometry Step

  28. 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

  29. Mass spectrometry Aebersold & Mann, Nature 2003

  30. Gavin et al. : 1167 baits 589 protein complexes (232 distinct) 3617 interactions among 1578 proteins Two large-scale mass spec experiments Gavin et al. Ho et al. Ho et al. : 725 baits

  31. Overlap in baits 3007 3419 198 1052 (454) 610 (493) 115 Overlap Reproducibility in AP/MS Gavin et al. : 1167 baits Ho et al. : 725 3225 interactions among 1440 proteins 3617 interactions among 1578 proteins

  32. 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

  33. Scoring protein-protein interactions

  34. Complex pull-downs Low confidence (rarely purified together) High confidence (often purified together) Topology based scoring of interactions Yeast two-hybrid D A B C High confidence (1 unshared interaction partners) Low confidence (4 unshared interaction partners) de Lichtenberg et al., Science 2005

  35. 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)

  36. 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)

  37. Filtering by subcellular localization de Lichtenberg et al., Science, 2005

More Related