Protein protein interactions

Introduction

Protein bunknown

Protein aknown

Interaction

Better understanding of protein b’s function

Introduction

Protein bFunction b

Protein aFunction a

Interaction

Detecting relationship between pathways

Detecting new pathways

Old world vs. new world

OLD WORLD

Discrete methods (1 X 1)

NEW WORLD

Comprehensive methods

MAINLY

IN THIS LECTURE

Yeast two hybrid systemSimple transcription

gene

Transcription

Complex

transcriptionDBD

DBD = DNA Binding Domain

AD

AD = Activation Domain

upstream activating sequence

UAS

Yeast two hybrid systemHybrid proteins

DBD

bait

AD

fish

Reporter gene

Transcription

Complex

transcription

UAS

DBD

bait

AD

fish

Yeast two hybrid systemHybrid proteins

Yeast two hybrid systemplasmids

Plasmid Apr

omot

erDBD bait

Plasmid B

prom

oter

AD fish

Yeast

Yeast two hybrid systemplasmids

Yeast

Report of the gene only in case of interaction

Between the two proteins

• Produce the yeast’s 6000 ORFs

Yeast array – producing the array

prom

oter

AD ORF

• Produce plasmids: each contains ORF + AD

• Transformation the plasmids into yeast cells

Yeast

• 2 colonies of each transformation are

inserted to the array

• ORF – Open Reading Frames

A comprehensive analysis of protein-protein interactions in S. cerevisiae P. Uetz et al..

A comprehensive analysis of protein-protein interactions in S. cerevisiae P. Uetz et al..

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

prom

oter

DBDORF

• Producing similar plasmids (DBD+protein)• Transformation the plasmids into yeast cells

YeastMATING & creatingdiploids

SELECTION OF LIVING COLONIES

BASED ON HIS3 PRODUCTION

DETECTING THE ARRAY’S PROTEIN

ACCORDING TO ITSPOSITION

Yeast array - results

BEFORE(Pcf11)

AFTER

RNA15

RNA14

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

Duplicates of a single DBD transformant

Activation Domain library

prom

oter

ADORF• Production of ORF+AD plasmids and transformants

prom

oter DBDORF

• Production of ORF+DBD plasmids and transformants

• Production of an AD library

The AD libraryMATING (haploids to diploids)Transferring to a selection plateDetecting the ORF’s using PCR

• 817 out of ~6000 proteins were detected as involved in protein-protein interactions

• total of 692 interactions were detected

Activation Domain library - results

Array vs. library - Comparison

ARRAYLIBRARY

TimeTakes a lot of timeRather quick

Amount of checked

interactions Plenty

(192X6000)Huge number

(6000X6000)Average number of

Interactions for an interacting protein3.31.8

Number of interactions of 12 proteins that were detected in both methods

4814

CONCLUSIONS - THE METHODS’

QUALITIES

QUICK, SIMPLER,

CHEAPER

SENSETIVE, BETTER

RESULTS

Protein arrays – producing the array

Producing the yeast’s 6000 ORF’s using plasmids

• Attaching histidine anchors to every protein

• Attaching the proteins to an array

Protein chips: from concept to practiceYoung-Sam Lee et al..

Protein chips: from concept to practiceYoung-Sam Lee et al..

Protein arrays – using the array

• Pouring a protein onto the array

Detecting the interactions

Using antibodiesthat detect the

interaction’s product

The poured Protein

is labeled

Sophisticatedassays

Mass Spectrometry of purified complexes

• Production of chimeric tagged proteins using plasmids

• The complex is isolated using the tag

• The protein creates a complex of proteins

• 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 MutationsWhat’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

MATING & creatingdiploids

STIMULATING THE CREATION OF SPORES

+SELECTIONFINDING THE DEAD

• Pour different yeasts carrying different mutations

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

ORTHOLOGS

CREATURE A

CREATURE B

Computational methods Genome analysis

• Interacting proteins have a tendency to be either present ot absent

together from fully sequenced genomes

• One gene in creature A = some genes in creature B

(ROSETTA STONE)

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