Transcript Discovery Research via in vivo Evolution

Discovery Research via
in vivo Evolution
Huang Lei, Tian He, Wen Ya, and Zhang Yi
Peking University, and
National Institute of Biological Sciences, Beijing
2008 03 02
Discovery Research in Biology
• To answer the question: ‘what’/‘whether’
• Example 1: what activates receptor X?
– Whether drug alpha activates X?
• Example 2: what suppresses gene Y?
– Whether gene beta suppresses Y?
• Example 3: what maintains stem cell state?
– Whether kinase gamma maintains stem cell
state?
Strategies for Discovery Research
• Two strategies towards the goal:
• Guess: answering whether, intelligent but
very few novel insight
• Screen: answering what, laboring but can
give anti-intuition insight
• Hereinafter we concentrate on screening
Complexity Theory for Screen
• You always have it in your first 100 lines or
you never have it -- Seymour Benzer on flies
• Complexity theory: when dimension grows,
for serial screening, complexity grows in
geometrical metrics
• Monte Carlo method complexity
• Simulated annealing: decelerating Monte
Carlo method
Example of Simulated Annealing in
Biological System
• Adaptive Immunity
Molecular components of adaptive
immunity
• Somatic hypermutation
Molecular components of adaptive
immunity
• DNA break and repair
AID at the center of adaptive
immunity
How does AID works?
C
•AID converts C to U, causing U:G mispairs.
•The mispairs are repaired through the base
excision repair (BER) or the mismatch repair
(MMR) pathways
•Mutations are introduced through the
intervention of translesion DNA polymerases.
U
UNG regulates
transition/transversion ratio
Limiting AID function
• Transcription rate of the target gene: AID
only targets ssDNA
• AID promoters and enhancers
• Epigenetic insulators
• Specific sequence bias
-Hotspots: DGYW/WRCH
(R = A/G, Y = T/C, W = A/T, D = A/G/T).
A Problem:
How to restrict AID function within the
targeted sequence? The genomic
damage must be avoided!
Possible solution:
Mimic the Immunoglobin structure?
in vivo evolution application based
on adaptive immunity
Problems (and solutions?)
• Mammalian cells grow slow
– Bacteria/yeast grow fast
• Mammalian cells are expensive
– Bacteria/yeast are cheap
• Eukaryote protein has to be correctly
folded and glycosylated
– Yeast better than bacteria?
AID can work in yeast
An Example
Class of drug target
Species
Number of
molecular
targets
Targets of approved drugs
Pathogen and human
324
Human genome targets of approved
drugs
Human
266
Targets of approved small-molecule
drugs
Pathogen and human
248
Targets of approved small-molecule
drugs
Human
207
Targets of approved oral small-molecule
drugs
Pathogen and human
227
Targets of approved oral small-molecule
drugs
Human
186
Targets of approved therapeutic
antibodies
Human
15
GPCR, deorphanization
and drug discovery
• GPCR: G protein coupled receptors
• A huge gene family
• Important pharmacological target
Sexual
Reproduction
in yeast
-- a GPCR signaling
pathway
How to get it done in yeast?
GPCR signaling mating pathway
expression of heterologous GPCRs
Four modification for heterogolous
GPCRs
 Introducing heterologous GPCRs
add a cleavable leader sequence to aid transport to the plasma
membrane
remove regions not required for interacting with the ligand or G
protein.
 Modifying the G protein
develop chimeric G alpha subunits to incorporate receptor binding
properties of mammalian subunits into a Gpa1 subunit that retains
efficient interaction with the yeast G beta gamma
Four modification for heterogolous
GPCRs
 Knockout some native genes and incorporating
reporter genes
knock out Ste2, Sst2, Far1
combine reporter genes behind PRE
 Autocrine system
establish an autocrine system
combine the ligand to a factor or alpha factor facilitating its secreting
but restrict on the membrane
What can we do with it ??
Our Plan …
Protocol…
Ade2
Peptide-alpha factor
IRES
His3
PRE
Ade2
lacO
IRES
lacZ
lacI
hAID
The whole system
One example using GPCR protocol for artificial evolution
No binding
between peptide
and GPCR
x
Signalling
Initially………………..
Peptide-alpha factor
Ade2
x
His3
PRE
Ade2
IRES
lacO
IRES
lacZ
lacI
hAID
No GPCR signalling: hAID is expressed to mutate peptide ligand
Binding between
peptide and
GPCR
Signalling
Until the peptide become an agonist of GPCR….
Ade2
Peptide-alpha factor
Fus1
IRES
His3
PRE
IRES
lacZ
lacI
x
Ade2
lacO
hAID
His3
GPCR is activated, AID is silenced…
lacZ
lacZ readout with fluorescence
……… or visual detection directly
Positive and negative selections
• Positive selection:
– his3 mediated histidine- survival
– High lacZ activity
• Negative selection:
– Raise in complete medium (let it grow!)
– Low or no lacZ activity
Applications for drug discovery
• Peptidergic ligand for specific GPCR
• Optimizing peptidergic ligand hits
• Finding conserved motif for
agonist/antagonist
Assay procedure: it is easy!
• Transform GPCR to ready-knockout lines
• Assay for constitutive activity
• Transform the peptide-encoding vector
library into a nice coupled GPCR line
• Grow the transformant in large vial with
evolution medium (His-, 3AT+)
• After sometime, collect the solution and
plate for colonies
• Sequence individual colony for hits
Further development on compound
structure
GPCR other than ligand
Taking the complexity of the GPCR
pathway into account
We can first use the simple yeast twoor three- hybrid systems for a test.
Yeast two-hybrid system
Peptide-Gal4-AD
Ade2
IRES
His3
UAS
Ade2
lacO
IRES
lacZ
lacI
hAID
For example,
the core circuit could be adopted into Y2H
The methods in two-hybrid systems
Generally, the cDNA encoding the DBD-X fusion
protein and the cDNA encoding the AD-Y fusion
protein are inserted into two plasmids, respectively,
and then both transformed into the yeast cells.
Sexual Reproduction
in yeast
Interaction mating methods can
also be used in two-hybrid systems.
The AD and DBD fusion proteins
begin in two different haploid yeast
strains with opposite mating types,
a and α, respectively.
To test for interaction,the hybrid
proteins are brought together by
mating, a process in which two
haploid cells fuse to form a single
diploid cell.
Further,
Yeast
three-hybrid
system
Application of Y3H
• RNA aptamer
screen
• … or: RNAinteracting
protein?
Applications other than GPCR
•
•
•
•
Nuclear receptor ligand screen
Protein interaction screen
Novel bacterial transcriptional biosensors?
Whatever you can think about! :)
Summary
• We present a simple core genetic circuit which
can evolve any desired target in vivo
• We present a unified, inexpensive solution for
both academical and industrial needs
• In vivo evolution brings greater capacity and
flexibility to screening
• Further assay development based on
mammalian systems such as immune cell lines
Acknowledgements
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Wang Yiping
Youri Pavlov
Rao lab members
iGEM 2007 members :)
PKU iGEM 2008 society :)