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C105A/C205A Uland Lau, Pardeep Singh, Joe Argus 4/17/12

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

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C105A/C205A Uland Lau, Pardeep Singh, Joe Argus 4/17/12

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  1. C105A/C205A Uland Lau, Pardeep Singh, Joe Argus 4/17/12

  2. 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: ?

  3. 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

  4. 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)

  5. “Single Ligand-Single Domain” System DD = destabilizing domain, POI = protein of interest

  6. 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)

  7. 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

  8. 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

  9. Characterization of Shld1-Responsive Destabilizing Domains

  10. 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.

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

  12. 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.

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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. PLoS ONE 7(1), 2012

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

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