Protein Interactions

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Protein Interactions

• David Wishart
• April, 2004

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PolyomX Efforts

• Microarrays
• Individual protein expression levels
• Proteomics
• Individual protein expression levels
• Tissue Arrays
• Individual protein expression levels
• SNPs
• Individual protein mutations

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The Protein Parts List
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However...

• This cataloging (which consumes most of
  bioinformatics) has been derogatively referred to
  as stamp collecting
• Having a collection of parts and names doesnt
  tell you how to put something together or how
  things connect -- this is biology

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Proteins Move in Pathways
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Proteins Assemble
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Proteins Interact
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Protein Interaction Tools and Techniques -
Experimental Methods

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3D Structure Determination

• X-ray crystallography
• grow crystal
• collect diffract. data
• calculate e- density
• trace chain
• NMR spectroscopy
• label protein
• collect NMR spectra
• assign spectra NOEs
• calculate structure using distance geom.

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Protein Interaction Domains
http//www.mshri.on.ca/pawson/domains.html
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Protein Interaction Domains
http//www.mshri.on.ca/pawson/domains.html
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Yeast Two-Hybrid Analysis

• Yeast two-hybrid experiments yield information on
  protein protein interactions
• GAL4 Binding Domain
• GAL4 Activation Domain
• X and Y are two proteins of interest
• If X Y interact then reporter gene is expressed

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Example of 2-Hybrid Analysis

• Uetz P. et al., A Comprehensive Analysis of
  Protein-Protein Interactions in Saccharomyces
  cerevisiae Nature 403623-627 (2000)
• High Throughput Yeast 2 Hybrid Analysis
• 957 putative interactions
• 1004 of 6000 predicted proteins involved

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Example of 2-Hybrid Analysis

• Rain JC. et al., The protein-protein interaction
  map of Helicobacter pylori Nature 409211-215
  (2001)
• High Throughput Yeast 2 Hybrid Analysis
• 261 H. pylori proteins scanned against genome
• gt1200 putative interactions identified
• Connects gt45 of the H. pylori proteome

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Another Way?

• Ho Y, Gruhler A, et al. Systematic identification
  of protein complexes in Saccharomyces cerevisiae
  by mass spectrometry. Nature 415180-183 (2002)
• High Throughput Mass Spectral Protein Complex
  Identification (HMS-PCI)
• 10 of yeast proteins used as bait
• 3617 associated proteins identified
• 3 fold higher sensitivity than yeast 2-hybrid

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Affinity Pull-down
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Protein Chips
Antibody Array Antigen Array
Ligand Array
Detection by SELDI MS, fluorescence, SPR,
electrochemical, radioactivity, microcantelever
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Protein (Antigen) Chips
H Zhu, J Klemic, S Chang, P Bertone, A Casamayor,
K Klemic, D Smith, M Gerstein, M Reed, M
Snyder (2000).Analysis of yeast protein kinases
using protein chips. Nature Genetics 26 283-289
ORF
GST
His6
Nickel coating
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Protein (Antigen) Chips
Nickel coating
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Arraying Process
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Probe with anti-GST Mab
Nickel coating
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Anti-GST Probe
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Probe with Cy3-labeled Calmodulin
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Functional Protein Array
Nickel coating
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Antigen Array (ELISA Chip)
Mezzasoma et al. Clinical Chem. 48121 (2002)
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Diagnostic Antigen Array
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Protein Chips
Antibody Array Antigen Array
Ligand Array
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Ciphergen Ligand Chips

• Hydrophobic (C8) Arrays
• Hydrophilic (SiO2) Arrays
• Anion exchange Arrays
• Cation exchange Arrays
• Immobilized Metal Affinity (NTA-nitroloacetic
  acid) Arrays
• Epoxy Surface (amine and thiol binding) Arrays

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Ciphergen ProteinChip
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Peptide/Protein Profile
E. coli
Salmonella
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Protein Interaction Tools and Techniques -
Computational Methods

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Sequence Searching Against Known Domains
http//www.mshri.on.ca/pawson/domains.html
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Motif Searching Using Known Motifs
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Text Mining

• Searching Medline or Pubmed for words or word
  combinations
• X binds to Y X interacts with Y X
  associates with Y etc. etc.
• Requires a list of known gene names or protein
  names for a given organism
• Sometimes called Textomy

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http//textomy.iit.nrc.ca/
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Pre-BIND

• Donaldson et al. BMC Bioinformatics 2003 411
• Used Support Vector Machine (SVM) to scan
  literature for protein interactions
• Precision, accuracy and recall of 92 for
  correctly classifying PI abstracts
• Estimated to capture 60 of all abstracted
  protein interactions for a given organism

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Rosetta Stone Method
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Interologs, Homologs, Paralogs...

• Homolog
• Common Ancestors
• Common 3D Structure
• Common Active Sites
• Ortholog
• Derived from Speciation
• Paralog
• Derived from Duplication
• Interolog
• Protein-Protein Interaction

YM2
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Finding Interologs

• If A and B interact in organism X, then if
  organism Y has a homolog of A (A) and a homolog
  of B (B) then A and B should interact too!
• Makes use of BLAST searches against entire
  proteome of well-studied organisms (yeast, E.
  coli)
• Requires list of known interacting partners

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A Flood of Data

• High throughput techniques are leading to more
  and more data on protein interactions
• This is where bioinformatics can play a key role
• Some suggest that this is the future for
  bioinformatics

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Interaction Databases

• BIND
• http//www.blueprint.org/bind/bind.php
• DIP
• http//dip.doe-mbi.ucla.edu/
• MINT
• http//mint.bio.uniroma2.it/mint/
• PathCalling
• http//portal.curagen.com/extpc/com.curagen.portal
  .servlet.Yeast

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The BIND Database

• BIND - Biomolecular Interaction Network Database
• Conceived and Developed by Chris Hogue, Tony
  Pawson, Francis Ouellette
• Designed to capture almost all interactions
  between biomolecules (large and small)
• Largest database of its kind

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BIND Data Model
E
S P
ES E-S
Interaction Record
P
S P
Chemical State Data
Chemical Action Data
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BIND Can Encode...

• Simple binary interactions
• Enzymes, substrates and conformational changes
• Restriction enzymes
• Limited proteolysis
• Phosphorylation (reversible)
• Glycosylation
• Intron splicing
• Transcriptional factors

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BIND
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BIND Query Result
click
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BIND Details
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BIND Details
click
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BIND Details
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DIP Database of Interacting Proteins
http//dip.doe-mbi.ucla.edu/
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DIP Query Page
CGPC
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DIP Results Page
click
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DIP Results Page
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MINT Molecular Interaction Database
http//mint.bio.uniroma2.it/mint/
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MINT Results
click
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KEGG Kyoto Encyclopedia of Genes and Genomes
http//www.genome.ad.jp/kegg/kegg2.html
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KEGG
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KEGG
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TRANSPATH
http//www.biobase.de/pages/products/transpath.htm
l
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BIOCARTA

• www.biocarta.com
• Go to Pathways
• Web interactive links to many signalling pathways
  and other eukaryotic protein-protein interactions

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Other Databases
http//www.hgmp.mrc.ac.uk/GenomeWeb/prot-interacti
on.html