Transcript Protein-protein Interaction Databases

Protein-protein Interactions
May 2, 2016
Why PPI?
• Protein-protein interactions determine outcome
of most cellular processes
• Proteins which are close homologues often
interact in the same way
• Protein-protein interactions place evolutionary
constraints on protein sequence and structural
divergence
• Pre-cursor to networks
PPI classification
• Strength of interaction
– Permanent or transient
• Specificity
• Location within polypeptide chain
• Similarity of partners
– Homo- or hetero-oligomers
• Direct (binary) or a complex
• Confidence score
Determining PPIs
• Small-scale methods
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Co-immunoprecipitation
Affinity chromatography
Pull-down assays
In vitro binding assays
• FRET, Biacore, AFM
– Structural (co-crystals)
PPIs by high-throughput methods
• Yeast two hybrid systems
• Affinity tag purification followed by mass
spectrometry
• Protein microarrays
• Microarrays/gene co-expression
– Implied functional PPIs
• Synthetic lethality
– Genetic interactions, implied functional PPIs
Yeast two hybrid system
Gal4 protein comprises DNA
binding and activating domains
Binding domain
interacts with
promoter
Activating domain
interacts with
polymerase
Measure reporter enzyme activity (e.g. blue colonies)
Yeast two hybrid system
•Gal4 protein: two domains do not need to be transcribed
in a single protein
•If they come into close enough proximity to interact,
they will activate the RNA polymerase
Two other protein domains (A & B) interact
Binding domain
interacts with
promoter
A
B
Activating domain
interacts with
polymerase
Measure reporter enzyme activity (e.g. blue colonies)
Yeast two hybrid system
• This is achieved using gene fusion
• Plasmids carrying different constructs can be expressed in
yeast
Binding domain as a translational
fusion with the gene encoding
another protein in one plasmid.
A
Activating domain as a
translational fusion with the gene
encoding a different protein in a
second plasmid.
B
If the two proteins interact, then GAL4 is expressed and blue colonies form
Yeast two hybrid
• Advantages
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Fairly simple, rapid and inexpensive
Requires no protein purification
No previous knowledge of proteins needed
Scalable to high-throughput
Is not limited to yeast proteins
• Limitations
– Works best with cytosolic proteins
– Tendency to produce false positives
Mass spectrometry
• Need to purify protein or protein complexes
• Use a affinity-tag system
• Need efficient method of recovering fusion protein in
low concentration
TAP (tandem affinity purification)
Spacer CBP
PCR product
TEV site
Protein A
Homologous
recombination
Chromosome
Fusion protein
Protein
Spacer CBP
TEV site
Protein A
Calmodulin
binding peptide
TAP process
"Taptag simple" by Chandres - Own work.
Licensed under CC BY-SA 3.0 via Wikimedia Commons
TAP
• Advantages
– No prior knowledge of complex composition
– Two-step purification increases specificity of pull-down
• Limitations
– Transient interactions may not survive 2 rounds of washing
– Tag may prevent interactions
– Tag may affect expression levels
– Works less efficiently in mammalian cells
Other tags
• HA, Flag and His
– Anti-tag antibodies can interfere with MS analysis
• Streptavidin binding peptide (SBP)
– High affinity for streptavidin beads
– 10-fold increase in efficiency of purification compared to
conventional TAP tag
– Successfully used to identify components of complexes in
the Wnt/b-catenin pathway
Used Dsh-2 and
Dsh-3 as bait
proteins
The KLHL12-Cullin-3 ubiquitin
ligase negatively regulates Wnt-bcatenin pathway by targeting
Dishevelled for degradation
Nature Cell Biology 4:348-357 (2006)
Binding partners of Bruton’s tyrosine kinase
Role in lymphocyte development &
B-cell maturation
Protein Science 20:140-149 (2011)
Databases of protein-protein interactions
• MINT – Molecular Interaction Database
– >240,000 interactions with 35,000 proteins
– Covers multiple species
• DIP -- Database of Interacting Proteins (UCLA)
– >79,000 interactions with >27,000 proteins
• CCSB – Proteomics base interactomes (Harvard)
– Human, viruses, C. elegans, S. cerevisiae
– Some unpublished data
• IntAct – EBI molecular interaction database
– Curated data from multiple sources
EBI IntAct
Submit single or lists of proteins
Provides method and reference for interactions
List format, can download easily
STRING database
• Search Tool for the Retrieval of Interacting Genes
– Integrates information from existing PPI data sources
– Provides confidence scoring of the interactions
– Periodically runs interaction prediction algorithms on
newly sequenced genomes
• v.10 covers >2000 organisms
http://string-db.org/
Networks in STRING database
Starting protein
• Nice graphical view
• Not so easy to
download lists of data
Networks can be expanded
3 indirect
interactions
Information about the proteins
Accessing Interaction data
• From a UniprotKB (reviewed record):
Transferring PPI annotation
• Most of the high-throughput PPI work is done in
model organisms
• Can you transfer that annotation a homologous
gene in a different organism?
Defining homologs
Orthologue of a protein is usually defined as the bestmatching homolog in another species
• Candidates with significant BLASTP E-value (<10-20)
• Having ≥80% of residues in both sequences
included in BLASTP alignment
• Having one candidate as the best-matching
homologue of the other candidate in corresponding
organism
Interologs
• If two proteins, A and B, interact in one organism and their
orthologs, A’ and B’, interact in another species, then the pair
of interactions A—B and A’—B’ are called interologs
• Align the homologs (A & A’, B & B’) to each other.
• Determine the percent identity and the E-value of both
alignments
• Then calculate the Joint identity and the Joint Evalue
Joint identity
J I = I AA' ´ I BB'
Joint E-value
J E = EAA' ´ EBB'
Transfer of annotation
• Compared interaction datasets between yeast, worm
and fly
• Assessed chance that two proteins interact with each
other based on their joint sequence identities
• Performed similar analysis based on joint E-values
– All protein pairs with JI ≥ 80% with a known interacting pair
will interact with each other
– More than half of protein pairs with JE  E-70 could be
experimentally verified.
Yu, H. et. al. (2004) Genome Res. 14: 1107-1118
PMID: 15173116
Examples of Protein-Protein Interologs
• In C. elegans, mpk-1 was experimentally shown to
interact with 26 other proteins (by yeast 2-hybrid)
• Ste5 is the homolog of Mpk-1 in S. cerevisiae
• Based on the similarity between the interaction
partners of mpk-1 and their closest homologs in S.
cerevisiae, the interolog approach predicted 5 of the
6 subunits of the Ste5 complex in S. cerevisiae
• This paper has been cited >100 times
• Why the interest in predicting protein-protein
interactions?
– Determining protein-protein interactions is challenging
and the high-throughput (genome-wide) methods are still
difficult and expensive to conduct
– Identifying candidate interaction partners for a targeted
pull-down assay is a more viable strategy for most labs
BIPS: BIANA Interolog Prediction Server
• Based on concept of
interolog
• Pre-defined
alignments
• Can submit list of
proteins to get
predicted interaction
partners
• Can filter predicted
list to increase
confidence
http://sbi.imim.es/web/index.php/research/servers/bips
Today in computer lab
• Finding PPIs in your sublist using IntAct
• Exploring a subset of PPIs using the STRING database
• Prediction of interactions homologs using the BIPS
server
• Continue Exercise 5 on protein analyses by exploring
possible PPI for your select genes