Transcript Protein-protein Interactions

Protein-protein Interactions
Hsueh-Fen Juan
2003, Mar 31
NTNU
Outline
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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
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
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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)
The Study of Protein-protein
Interaction by Mass Spectrometry
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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.
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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)
Yeast Two-hybrid
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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
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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
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UAS
HIS gene
Transfect yeast cells with genes encoding
hybrid 1 and hybrid 2
Transcriptioninitiation complex
HIS mRNA
Fishing for Proteins that
Interaction with Ras
ras gene
TRP
Bait plasmid
cDNA 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
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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)
Phage Display
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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
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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)
Protein-protein Interaction on
the Web
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DIP
Suiseki
Useful database
http://dip.doe-mbi.ucla.edu
DIP NEWS
January 24th, 2002
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Another release of the complete dataset. It
contains more than 11,000 individual proteinprotein 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 E2F transcription factor
•The talin/viniculin/actin system
Suiseki (2/3)
Suiseki (3/3)
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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)
Information Extraction (IE)
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A vast amount of data on proteinprotein 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
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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
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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
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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