Experimental and Computational Methods for Construction of Protein-Protein Interaction Map

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Experimental and Computational Methods for
Construction of Protein-Protein Interaction Map

• 2006.03.02
• ik-hyun BAe
• Molecular Genetic Lab
• Department of horticultural science

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Contents

• Introduction
• Experimental methods
• Computational methods
• Summary
• Reference

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Introduction

• Importance of protein interaction

Genome 30.000 genes Transcriptome
40-100.000 mRNAs Proteome
100-400.000 proteins Interactome
gt1.000.000 interactions
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Introduction

• Importance of protein interaction

• Protein-protein interaction are intrinsic to
  virtually every cellular process
• cell growth, cell cycle, metabolic pathway,
  signal transduction
• Understanding of how proteins function within
  the cell
• Gene mutation ? protein interaction confusion ?
  disease
• New drug development by protein function
  analysis
• Unknown protein may be discovered by known
  protein in protein signal pathway

(Benno Schwikowski, et al. 2000)
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I.Experimental methods

• In vitro

• Co-immunoprecipitation
• TAP-MS
• Far-western analysis
• GST-pull down assays
• Protein arrays

Bait Prey model

• In vivo

• Yeast two-hybrid system
• Phage display

Physical interaction between protein binding
domains
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Co-immunoprecipitation

• Immunoprecipitation (IP) experiment
• - immune response precipitation
• Affinity purify a bait protein antigen together
  with its binding partner using a specific
  antibody
• Capturing of immune complex by solid support
• Elution from the support and analysis by SDS-PAGE
  and detection by western blot

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Co-immunoprecipitation
(Eric Phizicky, et al. 1995)
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GST-pull down assays

• Affinity chromatography method
• Using a tagged or labeled bait by binding a
  specific affinity matrix
• Purification of a prey protein from a lysate
  sample or other protein-containing mixture
• GTH(glutathione)-GST(glutathione S-transferase)
  binding

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GST-pull down assays
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GST-pull down assays
Sepaharose bead-GTH(glutathione)
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TAP-MS (Tandem Affinity Purification-Mass
Spectrometry)

• Rapid purification of complexes without prior
  knowledge of the complex composition, activity,
  or function
• Ability to purify low abundant proteins/protein
  complexes
• Fusion of the TAP tag to the target protein
• Complex retrieval from tissue culture
• Large-scale studies

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TAP-MS (Tandem Affinity Purification-Mass
Spectrometry)
(Arnaud Droit, et al. 2005)
(Guillaume Rigaut, et al. 1999)
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Far-western analysis

• Similar strategy to Western blotting
• To determine receptor-ligand interactions and to
  screen libraries for interacting proteins
• Probe-a labeled or antibody-detectable bait
  protein
• Target protein-prey protein on the membrane
• Detection can be radioisotopic, chemiluminescent
  or colorimetric, depending on the probe label

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Far-western analysis
(Eric Phizicky, et al. 1995)
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Protein arrays

• Antibody-based or bait-based arrays
• High-throughput assays screening and detection
  of specific interactions of proteins from complex
  mixtures
• Protein expression profiling, protein-protein
  interaction and enzyme activity
• Binding between the capture proteins immobilized
  on a surface and the target proteins in the
  sample solution.

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Protein arrays
(Eric Phizicky, et al. 2003)
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Yeast two-hybrid system

• Detecting protein-protein interactions in yeast
• Transcriptional regulator system
• prey-bait model fusion proteins with a
  transcriptional activating domain (AD, prey), a
  DNA-binding domain (DBD, bait)
• Term two-hybrid derives from these two chimeric
  proteins.
• Most commonly used method for large scale,
  high-throughput identification of potential
  protein-protein interactions

Gene construction in yeast expression vectors
Two hybrid proteins bind
Expression of the reporter indicating that the
proteins bind
Forming a functional transcription activator
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Fishing with yeast two-hybrid system
Bait Protein X
Prey Unknown Protein
cDNA for X
tissue
Yeast plasmid expression vector
Total mRNA
Reverse transcriptase
cDNA
Transfection
Yeast plasmid expression vector
Transfection
Transformed yeast
Activation domain
Extract plasmids
Transfection
Positive yeast containing bait plus prey
Re-transformed yeast
Agar plate
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High-throughput Y2H screening
Two-hybrid SH3 domain protein-protein interaction
network
Principle of two-hybrid library and array
screens (Peter Uetz, et al. 2001)
(AH Yan Tong, et al. 2002)
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Phage display

• Molecular technique by which foreign proteins are
  expressed at the surface of phage particles
• Affinity selection of phage by binding to an
  antigen
• Elution of bound phage via acidic or enzymatic
  cleavage
• Reamplification of an enriched phage population.

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Phage display
Yeast SH3 domain protein-protein interaction
network
(AH Yan Tong, et al. 2002)
(William G.T. Willats. 2002)
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Comparison of each method
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II.Computational methods

• Bioinformatics method
• Statistical and simulational analysis through
  protein database and experimental data
• Finding missing components in protein pathway
• Reconstruction of signal pathway
• Application to comparative proteomics and
  evolution study of inter- or intra-species

Reconstruction of two metabolic pathways in E.
coli. (Arnaud Droit, et al, 2005)
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Epistasis analysis

• Influence of one locus on the expression of
  genetic variation at another locus
• Gene-Gene interaction
• Genetical epistasis Statistical
  epistasis!
• But Statistical epistasis Genetical
  epistasis?

(JH Moore, et al. 2005)
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Epistatic interaction in yeast metabolism

• Single and double knockout of 890 metabolic genes
• Epistatic effect buffering, aggravating,
  noninteracting
• Hierarchically organization of epistatic
  interaction network

Representation of the number of buffering and
aggravating interactions within and between
groups of genes
Buffering (green) and aggravating (red) gene
interaction network
(Daniel Segre, et al,
2005)
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Microarray analysis

• Standard statistical algorithms to arrange genes
• According to similarity in pattern of gene
  expression
• Coexpression of genes of known function with
  poorly characterized or novel genes
• A simple means of gaining the functions of many
  genes
• eQTL (expression quantitative trait loci)
  correlation analysis between trait and gene
  expression pattern

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Gene similarity metric S(X,Y)
N, i state of conditions G, X, Y
protein Goffset set to the mean of observations
on G FG standard deviation of G
eQTL analysis
(MICHAEL B. EISEN, et al. 1998)
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PSI PEI MAP

• PEIMAP (Protein Experimental Interactome MAP)
  using experimental protein interaction data
• PSIMAP (Protein Structural Interactome MAP)
  Interaction map using structural information
  about protein interactions
• - PDB (Protein Data Bank)
• - SCOP (Structural Classification of
  Proteins)

Jong Park, et al. (2001)
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Protein interaction database
(Xenarios I , et al, 2001)
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Summary

• Protein-Protein interaction map provides
  information about every cellular process.
• Development of new drug ,discovery of missing
  compounds in protein network and evolution study
• Experimental method have been established for
  monitoring protein interactions in vitro by
  protein fragment complementation and for
  screening protein interactions in vivo by cloning
  of interested genes.
• In vitro Co-immunoprecipitation, TAP-MS,
  Far-western analysis, GST-pull down assays,
  Protein arrays
• In vivo Yeast two-hybrid system, Phage display
• Computational methods are used to predict
  potential interactions, to validate the results
  of high-throughput interaction screens and to
  analyze the protein networks inferred from
  interaction databases.
• Epistasis and microarray analysis , PEI PSI map

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Reference

• Wan K. Kim, et al. Large-scale co-evolution
  analysis of protein structural interlogues using
  the global protein structural interactome map
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• Daeui Park, et al. Comparative interactomics
  analysis of protein family interaction networks
  using PSIMAP (protein structural interactome map)
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• Jong Park, et al. Mapping Protein Family
  Interactions Intramolecular and Intermolecular
  Protein Family Interaction Repertoires in the PDB
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• Peter Uetz, et al. Two-hybrid arrays. Current
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• Arnaud Droit, et al. Experimental and
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