Uncovering the Function of a Gene: Classical Genetics

1
Uncovering the Function of a GeneClassical
Genetics
In classical genetics, researchers generate
mutations, then work backwards to deduce the
normal function of the mutated gene Example -
mutate many Drosophila fruit flies - screen for
mutants that live unusually long lives -
identify gene mutated in the long-lived flies
(methusalah) - study how the normal version of
this gene shortens lifespan Drawback this
approach is not practical for mammals like us -
random mutations hard to generate and pinpoint -
many redundant copies of key genes - long
generation times, ethical considerations limit
experiments
2
Uncovering the Function of a GeneChemical
Genetics
In chemical genetics, researchers use small
molecules to disrupt the normal function of
protein targets, then identify those
targets Example - the compound colchicine
kills cells by blocking mitosis - radioactively
labeled colchicine bound to a protein in cells
that was later identified as tubulin - this is
how it was first discovered that microtubules
are polymers of a and b-tubulin Advantages
- small compounds can easily cross cell
membranes - can often be washed out to restore
normal phenotypes - can then serve as probes to
isolate the target proteins
3
Chemical Genetics
Use natural products or synthetic molecules to
induce a specific phenotype in whole
cells - - this approach has improved our
understanding of - intra-cellular signaling
pathways - cell cycle progression
- proteins involved in specific disease
states
4
Chemical Genetics
Thus, use small molecules as probes to link the
genome (which is information) to the proteome
(which carries out actions) - goal understand
the function of every protein Having a specific
inhibitor for every protein would give us great
control over whats going on in a cell - allow
specific modulation of the proteins contributing
to a particular disease state, for
instance
5
Identifying the Target for a Bioactive Molecule
Techniques for matching a small molecule to its
target (1) Affinity chromatography (2)
Photo-affinity chemical cross-linking (3)
Protein micro-arrays (4) mRNA-protein
fusions (5) Drug Westerns (6) Phage display
libraries
6
Affinity Chromatography
Small molecule is derivatized, linked to a solid
support - Column is loaded with
derivatized solid support - Incubated with
solubilized proteins (target binds to column)
- Washed with a buffer to rinse off unbound
proteins Bound protein is eluted from column by
washing with solution of free ligand -
Protein is then visualized by gel
electrophoresis (coomassie blue or silver
staining)
7
Affinity Chromatography
Carbodiimide coupling is a standard way to
covalently link molecules through carboxyl
and amine groups
8
Affinity Chromatography
How do you isolate a dopamine binding protein?
linked molecules
9
Photo-affinity Chemical cross-linking
Instead of linking drug to a solid support,
attach another molecule that is reactive with
light or protein functional groups (primary
amines) - This linker molecule will
covalently bind the protein once the
drug binds (non-covalently) to its protein
target - Linker may be radioactive, so the
protein gets labeled and can later be
visualized on a gel Process - Drug
linker complex enters cell drug binds to
target - Irradiated with light or allowed
to spontaneously react ?
10
Photo-affinity Chemical cross-linking
11
Photo-affinity Chemical cross-linking
Step 1 react drug with linker, in a
test tube
known drug

unknown protein
12
Photo-affinity Chemical cross-linking
Step 2 add drug-linker to cell - drug will bind
(non-covalently) to its protein target

13
Photo-affinity Chemical cross-linking
Step 3 shine light to activate photoreactive end
of the linker, which will
covalently bond to the protein
UV light
photoreactive end


14
Photo-affinity Chemical cross-linking
Step 3 shine light to activate photoreactive end
of the linker, which will
covalently bond to the protein - the
photoreactive end also carries a radioactive
label (), which now marks the protein

unknown protein is now radioactive, will show up
on film as a spot after being run out on a
protein gel
15
Protein Microarrays
Microarrays are tiny chips to which are attached
a large number of proteins ? proteins retain
their enzymatic functions, and bind ligands 2
kinds of microarrays (1) protein function
array Each protein in a cell is expressed
attached to a defined spot on chip - detects
which attached protein(s) the added ligands bind
to - by adding a drug attached to a fluorescent
marker, you can determine what cellular
protein(s) a labeled drug binds
16
Protein Microarrays
Microarrays are tiny chips to which are attached
a large number of proteins ? proteins retain
their enzymatic functions, and bind ligands 2
kinds of microarrays (2) protein-detecting
array Chip is coated with diverse
small-molecules, and washed with proteins to
see where binding occurs - detects which
attached drug a particular labeled protein binds
to
17
protein function array
yellow dots different bound proteins blue
dots different bound drugs
extract A red label extract B
green label
protein-detecting array
18
Making Microarrays I
- plain glass slides derivatized to yield a sheet
of maleimide groups - maleimide reacts with any
-SH group to form a covalent bond - from
combi-chem run, 1 bead placed in each well of a
microtitre plate
19
Making Microarrays II
- compound from each bead released, individually
spotted onto slide by robot (200 mm spots,
gt1,000 spots per cm2 on slide) - slide then
probed with labeled fluorescent protein(s) to
detect binding
20
MacBeath et al. 1999, PNAS 1217967
Trial run 3 different compounds with known
binding proteins spotted onto a slide, in
alternating fashion - then probed with all 3
proteins, each with a different color label -
each spot was correctly bound and labeled by its
cognate protein
21
Protein Microarrays Detection
How do you detect to which spot on a chip
proteins have bound? (1) Tag proteins with Green
Fluorescent Protein (GFP) - this will cause all
proteins to fluoresce under the right light ?
incubate chip with solution of GFP-tagged
cellular proteins - -
22
Protein Microarrays Detection
How do you detect to which spot on a chip
proteins have bound? (2) Surface plasmon
resonance - no protein modification is
necessary for detection Uses a laser as a highly
sensitive microbalance detects tiny mass
differences from the backside of the chip,
indicating which spots have proteins bound to
them Can be used in tandem with mass
spectrometry to detect binding events and
simultaneously determine the mass and the
sequence of the bound protein by MALDI-TOF MS
and MS/MS -- in a single experiment! - the
future of protein microarrays
23
Protein Microarrays Example
Kuruvilla et al. wanted to find a small molecule
inhibitor of a known protein, Ure2p (Nature
2002, 416 653-657) (1) Used diversity-oriented
synthesis to make a library of 3,780 small
molecules (2) Made a protein-detecting
microarray robotically spotted all
molecules onto a 4 cm2 glass slide (3) Probed
slide w/ fluorescently labeled Ure2p protein -
detected 8 spots, indicative of
protein-binding (4) 1 of 8 hits was found to
intensely inhibit Ure2p protein called
uretupamine
24
Protein Microarrays Example
Kuruvilla et al. wanted to find a small molecule
inhibitor of a known protein, Ure2p (5) Made
of series of derivatives of uretupamine, found 1
w/ improved inhibitory activity
(uretupamine B) (6) Used microarrays to probe
the effects of inhibiting Ure2p on overall
gene expression - discovered that only a subset
of the genes controlled by Ure2p protein are
expressed when Ure2p is inhibited by this
drug - showed that small molecules can provide
more information about multi-purpose proteins
than genetic deletions, by
selectively turning off some, but not all,
protein functions
25
Protein Microarrays Example
Process Kuruvilla et al. used a series of
methods we have discussed-- (1) combinatorial
chemistry (2) protein-detecting microarrays (3)
pharmacophore-based optimization -followed
by- (4) RNA-based microarrays classical
genetics, to explore effects of
Ure2p-inhibition on cellular physiology and gene
expression
26
Identifying the Target for a Bioactive Molecule
Techniques for matching a small molecule to its
target (1) Affinity chromatography (2)
Photo-affinity chemical cross-linking (3)
Protein micro-arrays (4) mRNA-protein
fusions (5) Drug Westerns (6) Phage display
libraries
Drawback
27
Identifying the Target for a Bioactive Molecule
Techniques for matching a small molecule to its
target (1) Affinity chromatography (2)
Photo-affinity chemical cross-linking (3)
Protein micro-arrays (4) mRNA-protein
fusions (5) Drug Westerns (6) Phage display
libraries
Advantage these methods link protein to its
gene sequence
28
mRNA-Protein fusions
Technique for physically linking mRNA transcript
to the end of each protein Attach the drug
puromycin to 3 end of all mRNA from a
cell Fusion proteins are made when ribosome
reaches 3' end of mRNA - Puromycin enters the
peptidyl transferase site - Creates a covalent
link between the mRNA and new protein Protein-mRN
A fusions can then be screened for protein
interactions using affinity chromatography or
other techniques - the mRNA of bound proteins
is reverse-transcribed and amplified by PCR
into a double-stranded DNA clone of the active
protein
29
3 end of mRNA is tagged with the drug
puromycin
Finished peptide ends up covalently bound to
end of puramycin-mRNA fusion
30
Drug Western
Combination of 2 widely used cell biology
methods 1) western blots proteins are
attached to nitrocellulose filters,
screened with antibodies 2) library screening
by colony lifts from plates of bacteria or phage
Protocol is akin to screening libraries with DNA
probes, changed to visualize protein-drug
interactions
31
1.
each colony is a bacterial clone containing a
cDNA insert it will produce large amounts of
its one protein (and each colony
likely has a different cDNA
insert, so will make a different protein)
2. blot onto a filter that will trap the
expressed proteins
32
1.
each colony is a bacterial clone containing a
cDNA insert it will produce large amounts of
its one protein (and each colony
likely has a different cDNA
insert, so will make a different protein)
3. wash with your drug, attached to
something visible (e.g., GFP)
2. blot onto a filter that will trap the
expressed proteins
4. go back to the plate and pick off the
colonies that produced binding proteins
33
Drug Western
Phage or bacterial cDNA library grown on agar
plates, covered by nitrocellulose filters
- each colony (spot on a plate) grew from a
single cell carrying a cDNA insert in a
plasmid (different gene cloned into each
colony) - soak filter in isopropyl
b-D-thiogalactopyranoside, which induces
expression of the inserted gene in each
individual colony Filters lifted from plates,
washed and hybridized with a chemical probe
covalently attached to a marker molecule that
visualizes binding Once a positive plaque or
colony is selected, the cDNA fragment
contained within is replicated, isolated and
sequenced ?
34
Drug Western
Example Identify binding protein of HMN-154
(anti-cancer drug w/ unknown mechanism of
action) HMN-154 linked to protein BSA, used to
screen colonies - antibodies to BSA used
to probe for binding Showed 2 proteins (NF-kB
and thymosin b-10) were binding targets of the
drug - confirmed by genetic knockout
techniques Tanaka et al. 1999
35
Phage Display Libraries
Create a library of cDNA sequences, with each
cDNA inserted into a different M13 bacteria
virus ( phage) - clone is positioned next
to DNA encoding virus coat protein P6 Virus then
transcribes a fusion protein linking the coat
protein to the unknown protein corresponding to
the cDNA insert - Fusion protein is placed
on the surface of the viral capsid (the
protein shell encasing the viral genome) In
other words, the attached protein encoded by the
cDNA clone is presented on the outer surface
of each phage particle
36
Phage Display Libraries
These virus particles are then used in affinity
chromatography - a drug has been
linked to a solid support - after
chromatography, the positive phage (those that
bind to the immobilized drug on the
column) are washed off - these phage, which
contain the clone of the binding protein, are
then amplified in bacteria Constraints
- protein must assume correct conformation on
phage surface - protein cannot inhibit
virus from exiting a bacterial cell -
attachment of unknown protein to P6 cannot block
binding site
37
(No Transcript)