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Biotech 2012 spring-6_protein_interactions_0
 

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    Biotech 2012 spring-6_protein_interactions_0 Biotech 2012 spring-6_protein_interactions_0 Presentation Transcript

    • Protein-protein Interactions
    • Outline  Introduction  The Study of Protein-protein Interaction by Mass Spectrometry  Yeast-Two-Hybrid  Phage Display  Protein-protein Interaction on the Web  Information Extraction (IE)
    •  Introduction  The Study of Protein-protein Interaction by Mass Spectrometry  Yeast-Two-Hybrid  Phage Display  Protein-protein Interaction on the Web  Information Extraction (IE)
    • Introduction Nature, 2000, 405:837-846.
    • Introduction  Intrinsic to every cellular process  Form the basis of phenomena -DNA replication and transcription -metabolism -signal transduction -cell cycle control
    •  Introduction  The Study of Protein-protein Interaction by Mass Spectrometry  Yeast-Two-Hybrid  Phage Display  Protein-Protein Interaction on the Web  Information Extraction (IE)
    • The Study of Protein-protein Interaction by Mass Spectrometry  A key question about a protein,in addition to when and where it is expressed, is with which other proteins does it interact. Interaction partners are an immediate lead into biological function and can potentially be exploited for therapeutic purposes. Creation of a protein– protein interaction map of the cell would be of immense value to understanding the biology of the cell. Purification of protein complex bait
    • The study of Protein-protein Interaction by Mass Spectrometry Nature Biotechnology, 1999, 17, 676-682.
    • The Study of Protein-protein Interaction by Mass Spectrometry Nature Biotechnology, 1999, 17, 676-682.
    • Generic Strategy to Analyze the Spatial Organization of Multi-protein Complexes by Cross-linking and Mass Spectrometry (1/2) Anal. Chem., 2000,72, 267-275.
    • Generic Strategy to Analyze the Spatial Organization of Multi-Protein Complexes by Cross-Linking and Mass Spectrometry (2/2) Anal. Chem., 2000,72, 267-275.
    •  Introduction  The Study of Protein-protein Interaction by Mass Spectrometry  Yeast-Two-Hybrid  Phage Display  Protein-Protein Interaction on the Web  Information Extraction (IE)
    • Yeast Two-hybrid  Researchers insert a gene in yeast for a "bait" protein alongside DNA for half of an "activator" protein. The other half of the activator DNA is then inserted alongside DNA for random "prey" proteins. The yeast cells are then grown up and the proteins are allowed to interact. If bait and prey proteins bind, the two halves of the activator protein be close enough to work together to turn on another yeast gene that turns the cell blue, signaling a match. ILLUSTRATION: C. SLAYDEN
    • Yeast Two-Hybrid System  Useful in the study of various interactions  The technology was originally developed during the late 1980's in the laboratory Dr. Stanley Fields (see Fields and Song, 1989, Nature).
    • Yeast Two-Hybrid Assay
    • Hybrid proteins DNA-binding domain Bait domain Activation domainFish domain Hybrid 1 Hybrid 2
    • Transcriptional Activation by Hybrid Protein in Yeast UAS HIS gene Transfect yeast cells with genes encoding hybrid 1 and hybrid 2 Transcription- initiation complex // HIS mRNA
    • Fishing for Proteins that Interaction with Ras ras gene TRP cDNA from library LEU Bait plasmid Fish plasmid Transfect into trp, leu, his mutant yeast cells Select for cells that grow in absence of tryptophan and leucine Plate selected cells on medium lacking histidine
    • Fishing for Proteins that Interaction with Ras HIS Ras hybrid Bait Fish Ras-interacting hybrid
    • Fishing for Proteins that Interaction with Ras Colony Formation
    • Fishing for Proteins that Interaction with Ras Ras hybrid Bait Fish Noninteracting hybrid HIS
    • Fishing for Proteins that Interaction with Ras No Colony Formation
    •  Introduction  The Study of Protein-protein Interaction by Mass Spectrometry  Yeast-Two-Hybrid  Phage Display  Protein-Protein Interaction on the Web  Information Extraction (IE)
    • Phage Display  A method where bacteriophage particles are made to express either a peptide or protein of interest fused to a capsid or coat protein.  It can be used for peptide epitopes, peptide ligands, enzyme substrates or single-chain antibody fragments.
    • Phage Display Bacterially expressed GST-fusion proteins or domains Immobilize individually into wells Wash unbound phage particles Amplify phage particles Sequence the cDNA insert
    •  Introduction  The Study of Protein-protein Interaction by Mass Spectrometry  Yeast-Two-Hybrid  Phage Display  Protein-Protein Interaction on the Web  Information Extraction (IE)
    • Protein-protein Interaction on the Web  DIP  Suiseki  Useful database
    • http://dip.doe-mbi.ucla.edu
    • DIP NEWS January 24th, 2002  Another release of the complete dataset. It contains more than 11,000 individual protein- protein interactions described in 1,800 research papers. Please, note that this is, most probably, the last dataset released in the old format as we anticipate the next one (to be released witin ~3-4 months; we'll try to prepare at least 3 updates a year) will be XML formatted.
    • Database of Interacting Proteins
    • Suiseki(1/3) •DNA replication •The Immune system •The E2F transcription factor •The talin/viniculin/actin system is a system for the extraction of protein- protein interactions from large collections of scientific text http://www.pdg.cnb.uam.es/suiseki/
    • Suiseki (2/3)
    • Suiseki (3/3)  Regulate  Activate
    •  Introduction  The Study of Protein-protein Interaction by Mass Spectrometry  Yeast-Two-Hybrid  Phage Display  Protein-protein Interaction on the Web  Information Extraction (IE)
    • Information Extraction (IE)  A vast amount of data on protein- protein interactions residues in the published literature, which never been entered into databases.  IE have been applied to gaining information on protein-protein interactions.
    • Mining Literature for Protein- protein Interactions
    • Mining Literature for Protein- protein Interactions
    • Mining Literature for Protein- protein Interactions
    • Detection of Protein Names  Protein Names are not standardized and are often used in different forms in free text style.
    • Creation of the Protein Name List Comp Funct Genom 2001, 2, 196-206
    • Extraction of the interactions  The nouns and verbs are taken from a hand constructed list containing nouns such as activation, phosphorylation or interaction, and verbs such as activates, binds or phosphorylates. Rules are applied directly to the text by string comparison. Comp Funct Genom 2001, 2, 196-206
    • Extraction of the Interactions  The sentence “The expressed p53 protein showed nuclear localization and its expression was associated with an induction of p21 and bax expression” relates p53 with p21 and bax but does not imply a physical interaction between them. Comp Funct Genom 2001, 2, 196-206
    • Distribution of the Protein Interaction Universe Comp Funct Genom 2001, 2, 196-206