C105A/C205A

Uland Lau, Pardeep Singh, Joe Argus

4/17/12

Our Genetic Toolkit

• “Modern experimental biology often relies on the perturbation of a gene followed by observation of the resulting phenotype to elucidate gene function.”

• Importance of perturbing a gene to determine its function– DNA level: cre/lox– Transcription level: tet/dox– mRNA level: RNAi– Protein level: ?

Modulating Genes at the Protein Level

• Small molecule specific inhibitors/activators– fast, dose-dependent, reversible– existence of small molecule inhibitor/activator, off-

target effects

• Shokat method– Fast, dose-dependent, reversible, specific, – Genetic work, limited to ATPases/GTPases

• Goal: develop a way to modulate any target protein’s activity rapidly, reversibly, and dose-dependently

Predecessors to a New Method

• Temperature sensitive “degron” in yeast– DHFRTS:X is stable at permissive temp at

degraded at high temp

• FKBP/MaRap/FRB system– Proteins FKBP and FRB only dimerize

when small molecule MaRap is present– Can use this to force colocalization of

target proteins X and Y (FKBP:X and FRB:Y)

“Single Ligand-Single Domain” System

DD = destabilizing domain, POI = protein of interest

Summary• Developed a “Single Ligand-Single Domain”

system for regulating the abundance of a target protein (in vivo) rapidly, reversibly, and tunably.

• Proofs of principle:– FKBP:YFP (N and C termini)– FKBP:YFP in multiple cell lines– FKBP:X (multiple different soluble proteins)– FKBP:CD8 (integral membrane protein)– Changing phenotype (morphology)

Cell-Based Screen

• Starting Point for DD: FKBP12 (F36V), called “FKBP” from now on

• Generate variants of FKBP (error-prone PCR), fuse to YFP and strong promoter

• Stably integrate constructs into fibroblasts• Treat with ligand, sort for YFP+• Remove ligand, sort for YFP-• Treat with ligand, sort for YFP+• Sequence constructs from remaining cells

Characterization of ligand-responsive destabilizing domains

• Five mutants were chosen (F15S, V24A, H25R, E60G, and L106P)

• Separately transduced into NIH3T3 fibroblast cells.

• A- Absence of Shld1 • B- Introduction of Shld1 treated with 3-fold dilutions of Shl1

(1microM-0.1 nM)• C-Varying dosages of Shld1• D-Treatment of Shld1 for 24 hours then washed to remove

Shld1 from media

Characterization of Shld1-Responsive Destabilizing Domains

E-Immunoblot of FKBP-YFP fusions from mock treatment(-) or treatment with 1 μM Shld1 for 24 hours.F-Proteasome inhibitor MG132 used in the presence or absence of Shld1.G-HeLa cells transfected with siRNA against lamin A/C monitored over 24 hours.

Fusion of an FKBP Destabilizing Domain to the N Terminus of YFP Results in Predictable and Reversible Small-Molecule Regulation of Intracellular Protein Levels.

Reversing the orientation of FKBP and YFP and determining the efficiency of these candidate destabilizing domains

-Overall: Destabilizing domains fused to the C terminus of YFP are less destabilizing than their N-terminal counterparts. Both domains respond similarly to Shld1.

Figure 4: FKBP Destabilizing Domains Confer Shld1-Dependent Stability to a Variety of Proteins

Regulation of a Membrane Protein• CD8α –

transmembrane glycoprotein – surface of T cells

• C terminus fused

• Suggests that the FKBP recruits cellular proteins for internalization of membrane proteins

Control of Cellular Phenotypes

• Expression of active small GTPases causes well-characterized changes in cell morphology

• Cdc42 – filopodia• RhoaA – stress

fibers• Arl7 – shrunken

cell phenotype

Conclusion and Discussion• Use of a synthetic small molecule (Shld-1) to

regulate the stability of specific proteins• Reliably control and predict the target protein’s

levels by dosage• Shown ligand-dependent stability in different

types of proteins (cytoplasmic, nuclear, and transmembrane) and various cell types

• Cell-permeable small molecules – ease of delivery– Fast, reversible, and tunable

• Probing protein function, physiological processes, and pathways

Comparison to RNAi

• Design of synthetic RNAi is difficult

• Effect of mRNA degradation can be variable

• Introducing into cells can be a challenge

• Typically, 48 hrs is needed for significant knockdown of protein levels

Disadvantages

• Requires making the fusion protein (gene knockin)

• Fusion protein needs to function like the native protein

• In-vivo experiments would be tedious– Need to introduce gene into animal– Regularly administer the ligand or small

molecule

PLoS ONE 7(1), 2012

Nature Medicine, Vol. 13, No. 5, 2007