Transcript The presentation part I

Protein protein
interactions
Introduction
Protein a
Protein b
known
unknown
Interaction
Better understanding of protein b’s function
Introduction
Protein a
Protein b
Function a
Function b
Interaction
Detecting new pathways
Detecting relationship
between pathways
Old world vs. new world
OLD WORLD
Discrete methods (1 X 1)
MAINL
Y
IN THIS
LECTURE
NEW WORLD
Comprehensive methods
Yeast two hybrid system
Simple transcription
AD = Activation Domain
DBD = DNA Binding Domain
AD
DBD
transcription
UAS
upstream activating sequence
gene
Yeast two hybrid system
Hybrid proteins
bait
DBD
AD
fish
Yeast two hybrid system
Hybrid proteins
AD
fish
bait
DBD
transcription
UAS
Reporter gene
Yeast two hybrid system
plasmids
Plasmid A
DBD
AD
Yeast
Plasmid B
Yeast two hybrid system
plasmids
Report of the gene only in case of interaction
Between the two proteins
Yeast
Yeast array – producing the array
A comprehensive analysis of protein-protein interactions in S. cerevisiae
P. Uetz et al..
• ORF – Open Reading Frames
• Produce the yeast’s 6000 ORFs
• Produce plasmids: each contains ORF + AD
• Transformation the plasmids into yeast cells
AD
• 2 colonies of each transformation are
inserted to the array
Yeast
Yeast array – producing the array
ORF of protein x+ AD
ORF of protein y+ AD
Yeast array – using the array
• Selection of 192 “easy” proteins
• Producing similar plasmids (DBD+protein)
• Transformation the plasmids into yeast cells
SELECTION OF
MATING
& creating
DETECTING
THE
LIVING COLONIES
ARRAY’S
diploids
BASED
ONPROTEIN
HIS3
ACCORDING
TO ITS
PRODUCTION
POSITION
DBD
Yeast
Yeast array - results
BEFORE
(Pcf11)
RNA14
AFTER
RNA15
Yeast array - Results over-view
• 2 undependable assays were preformed
for each of the 192 proteins.
• 87 out of 192 proteins were detected as
involved in protein-protein interactions
(passed the 2 assays)
• total of 281 interactions were detected
Activation Domain library
AD
• Production of ORF+AD plasmids
and transformants
DBD
• Production of ORF+DBD plasmids
and transformants
• Production of an AD library
Duplicates of a single
DBD transformant
to diploids)
The MATING
AD
library (haploids
Transferring
to a selection
plate
Detecting the ORF’s using PCR
Activation Domain library - results
• 817 out of ~6000 proteins were detected
as involved in protein-protein interactions
• total of 692 interactions were detected
Array vs. library - Comparison
ARRAY
LIBRARY
Takes a lot of time
Rather quick
Plenty
(192X6000)
Huge number
(6000X6000)
Average number of
Interactions for an interacting protein
3.3
1.8
Number of interactions of 12 proteins
that were detected in both methods
48
14
CONCLUSIONS THE METHODS’
QUALITIES
SENSETIVE,
BETTER
RESULTS
QUICK,
SIMPLER,
CHEAPER
Time
Amount of checked
interactions
Protein arrays – producing the array
Protein chips: from concept to practice
Young-Sam Lee et al..
Producing the yeast’s 6000 ORF’s using plasmids
• Attaching histidine anchors to every protein
• Attaching the proteins to an array
Protein arrays – using the array
• Pouring a protein onto the array
Detecting the interactions
The poured
Protein
is labeled
Using antibodies
that detect the
interaction’s product
Sophisticated
assays
Mass Spectrometry of purified complexes
• Production of chimeric tagged proteins using plasmids
• The protein creates a complex of proteins
• The complex is isolated using the tag
• The complex is separated using gel
electrophoresis
• Each protein is identified by Mass Spectrometry
Mass Spectrometry of purified complexes
BENEFITS
Identifying complex interactions
Reliability can be checked
DRAWBACKS
Needs specific conditions
Can lose loosely associated components
Tagging might disturb the “complexing”
Synthetic Lethal Mutations
What’s lethal mutation?
• Examine 2 genes , “viable”
and mutants
• Examining the creature carrying them
GENES
PR’ LEVELS
THE
CREATURE
Synthetic Lethal Mutations
Hypothesis : these proteins are in interactions
METHOD # 1: Synthetic Lethal Mutations
• Create an artificial DNA containing 2 genes with conditional mutations
• Change the conditions and detecting dead creatures
METHOD # 2: Synthetic Lethal Mutations Arrays
• Create a yeast array, every yeast contains a different mutation
• Pour different yeasts carrying different mutations
STIMULATING
THE
MATING
& creating
CREATION
OF
SPORES
diploids
FINDING
THE
DEAD
+ SELECTION
Computational methods
• Mentioned in this seminar, mainly for understanding proteins’
Functions and using to detect interactions
• Measuring mRNA levels under a variety of cellular conditions
• Grouping the genes that have similar transcriptional responses
IN VIVO, EXAMINE DIFFERENT CELL’S CONDITIONS
NOT SO ACCURATE
Computational methods
Genome analysis – IN SILICO
• Genes that are consistently in the same operon, in the same order
but in different and distanced creatures
CREATURE A
ORTHOLOGS
CREATURE B
Computational methods
Genome analysis
• Interacting proteins have a tendency to be either present ot absent
together from fully sequenced genomes
(ROSETTA STONE)
• One gene in creature A = some genes in creature B
CREATURE A
CREATURE B
1 polypeptid
3 polypeptids
Computational methods
Genome analysis
Inexpensive, fast, “widened” with the genomes DB
Otology relationships are not so clear,
not always reliable