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 –DHFR TS :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