Related Experiment Videos
Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid
S D Liberles1, S T Diver, D J Austin
1Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Abstract:
The natural product rapamycin has been used to provide temporal and quantitative control of gene expression in animals through its ability to interact with two proteins simultaneously. A shortcoming of this approach is that rapamycin is an inhibitor of cell proliferation, the result of binding to FKBP12-rapamycin-associated protein (FRAP). To overcome this limitation, nontoxic derivatives of rapamycin bearing bulky substituents at its C16-position were synthesized, each in a single step. The isosteric isopropoxy and methallyl substituents with the nonnatural C16-configuration abolish both binding to FRAP and inhibition of T cell proliferation. Binding proteins for these derivatives were identified from libraries of cDNAs encoding mutants of the FKBP12-rapamycin-binding (FRB) domain of FRAP by using a mammalian three-hybrid transcription assay. Targeting of the mutations was guided by the structure of the FKBP12-rapamycin-FRB ternary complex. Three compensatory mutations in the FRB domain, all along one face of an alpha-helix in a rapamycin-binding pocket, were identified that together restore binding of the rapamycin derivatives. Using this mutant FRB domain, one of the nontoxic rapamycin derivatives induced targeted gene expression in Jurkat T cells with an EC50 below 10 nM. Another derivative was used to recruit a cytosolic protein to the plasma membrane, mimicking a process involved in many signaling pathways.
Insights
Researchers developed non-toxic rapamycin derivatives for controlled gene expression. These compounds avoid inhibiting cell proliferation, enabling safer applications in biological research and signaling pathway studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Rapamycin controls gene expression by binding to FKBP12-rapamycin-associated protein (FRAP).
- Rapamycin's inhibition of cell proliferation limits its use.
- FKBP12-rapamycin-binding (FRB) domain is crucial for rapamycin's function.
Purpose of the Study:
- To synthesize non-toxic rapamycin derivatives that do not inhibit cell proliferation.
- To identify binding proteins for these novel rapamycin derivatives.
- To demonstrate the utility of these derivatives in targeted gene expression and protein recruitment.
Main Methods:
- Synthesis of rapamycin derivatives with bulky C16 substituents.
- Mammalian three-hybrid transcription assay to identify binding proteins.
- Structure-guided mutagenesis of the FRB domain.
- Assays for T cell proliferation inhibition and gene expression induction.
Main Results:
- Non-toxic rapamycin derivatives were synthesized, abolishing FRAP binding and T cell proliferation inhibition.
- Compensatory mutations in the FRB domain restored binding to the novel derivatives.
- One derivative induced targeted gene expression in Jurkat T cells with high potency (EC50 < 10 nM).
- Another derivative facilitated cytosolic protein recruitment to the plasma membrane.
Conclusions:
- Non-toxic rapamycin derivatives offer improved control over gene expression without cell proliferation inhibition.
- Mutant FRB domains can be engineered to bind specific rapamycin analogs.
- These derivatives represent valuable tools for studying biological pathways and gene regulation.