Uncovering the Function of a Gene: Classical Genetics

Uncovering the Function of a Gene:Classical 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

Uncovering the Function of a Gene:Chemical 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 and -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

Chemical GeneticsUse 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

Chemical GeneticsThus, 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 what’s going on in a cell

- allow specific modulation of the proteins contributing to a particular disease state, for instance

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

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)


Carbodiimide coupling is a standard way to covalently link molecules through carboxyl and amine groups


linked molecules

How do you isolate a dopamine binding protein?

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



Step 1: react drug with linker, in a test tube

unknown protein

known drug

*


Step 2: add drug-linker to cell

- drug will bind (non-covalently) to its protein target

*


Step 3: shine light to activate photoreactive end of the linker, which will covalently bond to the protein

UV lightphotoreactive end

*

*


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

*

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

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

protein function array

yellow dots:differentboundproteins

blue dots:differentbounddrugs

protein-detecting array

extract A = red label

extract B = green label

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

Making Microarrays II

- compound from each bead released, individually spotted onto slide by robot (200 m spots, >1,000 spots per cm2 on slide)

- slide then probed with labeled fluorescent protein(s) to detect binding

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

MacBeath et al. 1999, PNAS 121:7967

Protein Microarrays: Detection

How do you detect to which spot on a chip proteins have bound?

(1) Tag proteins with Green Fluorescent ProteinGreen Fluorescent Protein (GFP)

- this will cause all proteins to fluoresce under the right light

incubate chip with solution of GFP-tagged cellular proteins

-

-

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

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


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


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







(5) Drug Westerns


Drawback:







(5) Drug Westerns


Advantage: these methods link protein to its gene sequence

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-mRNA 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

3’ end of mRNA is tagged with the drug puromycin

Finished peptide ends up covalently bound to end of puramycin-mRNA fusion

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

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

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

1.

3. wash with your drug, attached to something visible (e.g., GFP)

4. go back to the plate and pick off the colonies that produced binding proteins

Drug WesternPhage 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 -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

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-B and thymosin -10) were binding targets of the drug - confirmed by genetic knockout techniques

Tanaka et al. 1999

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

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

Documents

Uncovering the Function of a Gene: Classical Genetics