Protein-protein Interactions

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Protein-protein Interactions
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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)

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• Introduction
• The Study of Protein-protein Interaction by Mass
  Spectrometry
• Yeast-Two-Hybrid
• Phage Display
• Protein-protein Interaction on the Web
• Information Extraction (IE)

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Introduction
Nature, 2000, 405837-846.
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Introduction

• Intrinsic to every cellular process
• Form the basis of phenomena
• -DNA replication and transcription
• -metabolism
• -signal transduction
• -cell cycle control

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• Introduction
• The Study of Protein-protein Interaction by Mass
  Spectrometry
• Yeast-Two-Hybrid
• Phage Display
• Protein-Protein Interaction on the Web
• Information Extraction (IE)

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The Study of Protein-protein Interaction by Mass
Spectrometry
bait

• 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
  proteinprotein interaction map of the cell would
  be of immense value to understanding the biology
  of the cell.

Purification of protein complex
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The study of Protein-protein Interaction by Mass
Spectrometry

• Nature Biotechnology, 1999, 17, 676-682.

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The Study of Protein-protein Interaction by Mass
Spectrometry
Nature Biotechnology, 1999, 17, 676-682.
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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.
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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.
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• Introduction
• The Study of Protein-protein Interaction by Mass
  Spectrometry
• Yeast-Two-Hybrid
• Phage Display
• Protein-Protein Interaction on the Web
• Information Extraction (IE)

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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

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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).

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Yeast Two-Hybrid Assay
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Hybrid proteins
DNA-binding domain
Bait domain
Activation domain
Fish domain
Hybrid 1
Hybrid 2
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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
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Fishing for Proteins that Interaction with Ras
ras gene
cDNA from library
TRP
LEU
Fish plasmid
Bait 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
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Fishing for Proteins that Interaction with Ras
Ras hybrid
Bait
Fish
Ras-interacting hybrid
HIS
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Fishing for Proteins that Interaction with Ras
Colony Formation
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Fishing for Proteins that Interaction with Ras
Ras hybrid
Bait
Fish
Noninteracting hybrid
HIS
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Fishing for Proteins that Interaction with Ras
No Colony Formation
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• Introduction
• The Study of Protein-protein Interaction by Mass
  Spectrometry
• Yeast-Two-Hybrid
• Phage Display
• Protein-Protein Interaction on the Web
• Information Extraction (IE)

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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.

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Phage Display

• Bacterially expressed GST-fusion proteins or
  domains

Immobilize individually into wells
Wash unbound phage particles
Amplify phage particles
Sequence the cDNA insert
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• Introduction
• The Study of Protein-protein Interaction by Mass
  Spectrometry
• Yeast-Two-Hybrid
• Phage Display
• Protein-Protein Interaction on the Web
• Information Extraction (IE)

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Protein-protein Interaction on the Web

• DIP
• Suiseki
• Useful database

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http//dip.doe-mbi.ucla.edu
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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.

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Database of Interacting Proteins
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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 E2F transcription factor
• The talin/viniculin/actin system

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Suiseki (2/3)
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Suiseki (3/3)

• Regulate
• Activate

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• Introduction
• The Study of Protein-protein Interaction by Mass
  Spectrometry
• Yeast-Two-Hybrid
• Phage Display
• Protein-protein Interaction on the Web
• Information Extraction (IE)

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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.

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Mining Literature for Protein-protein Interactions
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Mining Literature for Protein-protein Interactions
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Mining Literature for Protein-protein Interactions
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Detection of Protein Names

• Protein Names are not standardized and are often
  used in different forms in free text style.

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Creation of the Protein Name List
Comp Funct Genom 2001, 2, 196-206
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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
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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
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Distribution of the Protein Interaction Universe
Comp Funct Genom 2001, 2, 196-206