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Controlling programmed cell death with a cyclophilin-cyclosporin-based chemical inducer of dimerization
P J Belshaw1, D M Spencer, G R Crabtree
1Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA. sls@slsiris.harvard.edu
Background:
Cell death can occur either from physical damage (necrosis) or cellular suicide (apoptosis). Apoptosis is essential for the development of multicellular organisms and disregulated apoptosis underlies many human diseases. The Fas receptor (Fas) is a membrane signaling protein that mediates a death signal following its aggregation by the Fas ligand. We have described methods to induce the association of proteins using cell-permeable molecules called chemical inducers of dimerization (CIDs). Here we describe the synthesis of a novel CID, (CsA)2, that has two identical protein-binding surfaces derived from the immunosuppressant cyclosporin A (CsA). We use this CID to deliver a death signal to cells expressing a fusion protein containing cyclophilin (CyP, the protein receptor for cyclosporin) and the cytoplasmic signaling domain of Fas.
Results:
(CsA)2 was synthesized in six synthetic steps and 30% overall yield from cyclosporin. It binds to two CyP proteins simultaneously, but does not inhibit T-cell signaling, presumably because the (CsA)2-CyP complex does not bind to calcineurin. Jurkat cells stably transfected with constructs encoding myristoylated CyP-Fas fusion proteins undergo apoptosis in response to nanomolar quantities of (CsA)2. Constructs containing a mutation in the myristoylation signal are defective for signaling.
Conclusions:
The Fas signaling pathway can be activated with a cell-permeable CID derived from CsA in cells expressing an appropriately engineered Fas construct, which must be localized at the membrane. This new class of homodimerizing CIDs will be useful for in-depth analysis of protein association events in complex systems, including transgenic animals. Now that several CIDs with distinct dimerization characteristics are available, it should be possible to induce the activation of multiple pathways with complete specificity.
Insights
Researchers developed a novel chemical inducer of dimerization, (CsA)2, derived from cyclosporin A. This compound effectively triggers apoptosis in cells expressing a specific Fas fusion protein, offering a new tool for studying cell death pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Apoptosis, or programmed cell death, is crucial for development and implicated in diseases.
- The Fas receptor mediates cell death signals upon aggregation.
- Chemical inducers of dimerization (CIDs) can induce protein association.
Purpose of the Study:
- To synthesize and characterize a novel CID, (CsA)2, derived from cyclosporin A.
- To investigate the ability of (CsA)2 to induce Fas-mediated apoptosis.
- To explore the utility of (CsA)2 for studying protein-protein interactions.
Main Methods:
- Synthesis of (CsA)2 from cyclosporin A.
- Co-expression of cyclophilin-Fas fusion proteins in Jurkat cells.
- Treatment of cells with (CsA)2 to induce apoptosis.
Main Results:
- (CsA)2 was synthesized with 30% overall yield.
- (CsA)2 induced apoptosis in Jurkat cells expressing myristoylated cyclophilin-Fas fusion proteins at nanomolar concentrations.
- Mutations in the myristoylation signal abolished apoptosis induction.
Conclusions:
- Cell-permeable CIDs derived from CsA can activate the Fas pathway in engineered cells.
- (CsA)2 represents a new class of homodimerizing CIDs for analyzing protein association.
- This approach enables specific induction of multiple signaling pathways.