Proteinprotein Interactions HsuehFen Juan 2003 Mar 31 NTNU
- Slides: 51
Protein-protein Interactions Hsueh-Fen Juan 2003, Mar 31 NTNU
Outline n n n Introduction The Study of Protein-protein Interaction by Mass Spectrometry Yeast-Two-Hybrid Phage Display Protein-protein Interaction on the Web Information Extraction (IE)
n n n 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 n n Intrinsic to every cellular process Form the basis of phenomena -DNA replication and transcription -metabolism -signal transduction -cell cycle control
n n n 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 n 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. bait Purification of protein complex
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.
n n n 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 n 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 n n 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 Hybrid 1 Fish domain Activation domain Hybrid 2
Transcriptional Activation by Hybrid Protein in Yeast UAS // HIS gene Transfect yeast cells with genes encoding hybrid 1 and hybrid 2 Transcriptioninitiation complex HIS m. RNA
Fishing for Proteins that Interaction with Ras ras gene TRP Bait plasmid c. DNA from library LEU 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 Bait Ras hybrid Fish Ras-interacting hybrid HIS
Fishing for Proteins that Interaction with Ras Colony Formation
Fishing for Proteins that Interaction with Ras Bait Ras hybrid Fish Noninteracting hybrid HIS
Fishing for Proteins that Interaction with Ras No Colony Formation
n n n 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 n n 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 c. DNA insert
n n n 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 n n n DIP Suiseki Useful database
http: //dip. doe-mbi. ucla. edu
DIP NEWS January 24 th, 2002 n Another release of the complete dataset. It contains more than 11, 000 individual 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) http: //www. pdg. cnb. uam. es/suiseki/ is a system for the extraction of protein -protein interactions from large collections of scientific text • DNA replication • The Immune system • The E 2 F transcription factor • The talin/viniculin/actin system
Suiseki (2/3)
Suiseki (3/3) n n Regulate Activate
n n n 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) n n A vast amount of data on 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 Proteinprotein Interactions
Mining Literature for Proteinprotein Interactions
Mining Literature for Proteinprotein Interactions
Detection of Protein Names n 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 n 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 n The sentence “The expressed p 53 protein showed nuclear localization and its expression was associated with an induction of p 21 and bax expression” relates p 53 with p 21 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
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