Biotech 2012 spring-6_protein_interactions_0

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



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


Interaction by Mass Spectrometry

Yeast-Two-Hybrid Phage Display Protein-Protein Interaction on the


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.


Interaction by Mass Spectrometry Yeast-Two-Hybrid Phage Display Protein-Protein Interaction on the


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 geneTransfect 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


HIS

Ras hybridBait

Fish

Ras-interacting hybrid


Colony Formation


Ras hybridBait

Fish

Noninteracting hybrid

HIS


No Colony Formation


Interaction by Mass Spectrometry Yeast-Two-Hybrid Phage Display Protein-Protein Interaction on the


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 DisplayBacterially expressed GST-fusion proteins or domains

Immobilize individually into wells

Wash unbound phage particles

Amplify phage particles

Sequence the cDNA insert


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 NEWSJanuary 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

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

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.


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.


Distribution of the Protein Interaction Universe


Biotech 2012 spring-6_protein_interactions_0

Technology

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