Protein Interactions

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Protein Interactions

• Michel Dumontier, Ph.D.
• Carleton University
• michel_at_bioinfocg.com

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Outline

• Protein interactions
• Discovery
• Experimental
• Storage

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Molecular Interactions

• Between two molecular objects
• DNA, RNA, gene, protein, molecular complex, small
  molecule, photon
• Binding Sites
• Under some Experimental Condition
• With a particular Cellular Location
• Possibly having a Chemical Action

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Interaction Discovery

• Databases
• Fully electronic
• Easily computer readable
• Literature
• Increasingly electronic
• Human readable
• Biologists brains
• Richest data source
• Limited bandwidth access
• Experiments
• Basis for models

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Yeast Two Hybrid Assay

• The two-hybrid system is a molecular genetic tool
  which facilitates the study of protein-protein
  interactions.
• If two proteins interact, then a reporter gene is
  transcriptionally activated.
• e.g. gal1-lacZ - the beta-galactosidase gene
• A colour reaction can be seen on specific media.
• You can use this to
• Study the interaction between two proteins which
  you expect to interact
• Find proteins (prey) which interact with a
  protein you have already (bait).

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Two-hybrid assay
SNF4
1.
B
SNF1
3.
A
2.
GAL4-DBD
Transcription activation domain
UASG
4.
Fields S. Song O. Nature. 1989 Jul
20340(6230)245-6. PMID 2547163
GAL1
Allows growth on galactose
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Some Two-hybrid caveats
1.
3.
A
2.
4.
Does the DNA Binding Domain fusion have activity
by itself?
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Some Two-hybrid caveats
1.
B
C
3.
A
2.
4.
Is the interaction mediated by some other
protein?
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Some Two-hybrid questions
1.
B
3.
A
2.
Are the proteins expresssed?Are they
over-expressed?Are they in-frame?Are the
interacting domains defined?Was the observation
reproducible?Was the strength of interaction
significant?Was another method used to back-up
the conclusion? Are the two proteins from the
same compartment?
4.
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Affinity purification
A
this molecule will bind the tag.
tag modification(e.g. HA/GST/His)
Protein of interest
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Affinity purification
the cell
A
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Affinity purification
lots of other untagged proteins
the cell
A
B
naturally binding protein
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Affinity purification
Ruptured membranes
A
B
cell extract
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Affinity purification
A
B
untagged proteinsgo through fastest(flow-through
)
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Affinity purification
A
B
tagged complexes are slower and come out later
(eluate)
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Some affinity purification questions
Is the bait protein expressed and in frame? Is
the bait protein observed?Is the bait protein
over-expressed?Are the interacting domains
defined?Was the observation reproducible?Was
the interactor found in the background?Was the
strength of interaction significant? Was the
interaction saturable? Was the interactor
stoichiometric with the bait protein?Was another
method used to back-up the conclusion?Was
tandem-affinity purification (TAP) used? Was the
interaction shown using an extract or a purified
protein? Is the inverse interaction
observable? Are the two proteins from the same
compartment? Are the two proteins known to be
involved in the same process? Is the interactor
likely to be physiologically significant?
A
B
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Some affinity purification caveats
First and most importantly, this is only a
representation of the observation. You can only
tell what proteins are in the eluate you cant
tell how they are connected to one another. If
there is only one other protein present (B), then
its likely that A and B are directly
interacting. But, what if I told you that
two other proteins (B and C) were present along
with A.
A
B
A
C
B
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Complexes with unknown topology
A
A
A
B
C
B
C
B
C
Which of these models is correct? The complex
described by this experimental result is said to
have an Unknown Topology.
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Complexes with unknown stoichiometry
A
A
B
C
Heres another possibility? The complex described
by this experimental result is also said to have
Unknown Stoichiometry.
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High-throughput Mass Spectrometric Protein
Complex Identification (HMS-PCI)
Mike Tyers, SLRI
Ste12
Ho et al. Nature. 2002 Jan 10415(6868)180-3
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Synthetic Genetic Interactions

• Synthetic genetic interactions (lethal, slow
  growth)
• Mate two mutants without phenotypes to get a
  daughter cell with a phenotype
• Synthetic lethal (SL), slow growth
• robotic mating using the yeast deletion library
• Genetic interactions provide functional data on
  protein interactions or redundant genes
• About 23 of known SLs (1295 - YPDMIPS) are
  known protein interactions in yeast

Tong et al. Science. 2001 Dec 14294(5550)2364-8
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Working overtimeCharlie Boones Robots
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Synthetic Genetic Interactions in Yeast
Tong, Boone
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SGA Synthetic Genetic Interaction Network 2004
1000 Genes4000 Interactions132 SGA Screens
Tong, Boone, Science, Feb 2004
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A measure of confidence?

• How do you know if the interaction really exists?
  
• Each method has its advantages and disadvantages.
  
• Be aware of systematic errors (i.e. tag effects)
• Be aware of contaminating proteins.
• Each method observes interactions from a slightly
  different experimental condition.
• Support from many different sources is certainly
  better than just one.

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Outline

• Molecular interactions
• Discovery
• Storage
• Databases
• File Formats
• Data Mining

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Interaction/Pathway Databases

• Arguably the most accessible data source, but...
• Varied formats, representation, coverage
• Pathway data extremely difficult to combine and
  use

Pathway Resource List (http//cbio.mskcc.org/prl/)
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http//bind.ca

• A free, open-source database for archiving and
  exchanging molecular assembly information. BIND
  is managed by the Blueprint Initiative at Mount
  Sinai Hospital in Toronto.
• The database contains
• Interactions/Reactions
• Molecular complexes
• Pathways
• BIND has an extensive data model, GNU software
  tools and is based on the NCBI toolkit extended
  recently to XML/Java
• The 175000 BIND records are curated and
  validated.

Bader GD, Betel D, Hogue CW. (2003) BIND the
Biomolecular Interaction Network Database.
Nucleic Acids Res. 31(1)248-50 PMID 12519993
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BIND Interaction Types
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Interaction Experimental Evidence in BIND
Remaining1
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55 Identifier Searches Supported!
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GI Pair - CSV Export
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BIND Record Header

• BIND record identifier
• Description Division
• Publications that support or dispute interaction
• Export Options
• Network Visualization

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BIND Record View
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BIND Record View

• The Interacting Molecules (A and B)
• Main identifier GI
• Organism
• Cross-references and aliases
• Gene Ontology terms
• Proteoglyphs
• Graphical representations of domain and protein
  structure.
• Ontoglyphs
• Graphical representations of molecule function,
  localization and binding

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Gene Ontology

• Functional protein annotation
• http//www.geneontology.org
• Controlled vocabulary for protein function and
  localization
• Molecular function e.g. DNA helicase
• Biological process e.g. mitosis
• Cellular Component e.g. nucleus
• Thousands of terms

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Ontoglyph Summary View
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Ontoglyph Filtering
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Other Interaction Databases

• DIP
• http//dip.doe-mbi.ucla.edu
• MINT
• http//mint.bio.uniroma2.it/mint
• MIPS
• http//mips.gsf.de/proj/yeast/tables/interaction/
  
• IntAct EBIs interaction database
• http//www.ebi.ac.uk/intact/
• Human Protein Interaction Database
• http//www.hpid.org/
• TRANSFAC transcription factors
• http//www.gene-regulation.com/

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Information Exchange
With Data Exchange Format
gt100 DBs and tools Tower of Babel
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Data Exchange File Formats

• BIND http//bind.ca
• Peer reviewed but closed process (Spec v3.1)
• ASN.1 or XML DTD/Schema
• PSI-MI http//psidev.sourceforge.net
• Peer reviewed, HUPO community standard
• Widely adopted
• BioPax http//www.biopax.org
• Community schema (Sloan Kettering, BioPathways
  Consortium)
• XML Schema, OWL, Protégé and GKB
• SBML
• Widely adopted for representing models of
  biochemical reaction networks

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BIND
ASN.1 (text)
XML
Flat File
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PSI level 2
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PSI Record Format
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BioPAX
http//www.biopax.org

• Represent
• Metabolic pathways
• Signaling pathways
• Protein-protein, molecular interactions
• Gene regulatory pathways
• Genetic interactions
• Accommodate representations used in existing
  databases such as BioCyc, BIND, WIT, aMAZE, KEGG,
  Reactome, etc.
• Community effort (open meetings)

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Conclusion

• Many experimental techniques to generate
  interaction data
• Interaction databases like BIND are a great
  resource for building up interaction networks
  into pathways
• Common standards for file formats imperative for
  making use of all this data!