Controlling programmed cell death with a cyclophilin-cyclosporin-based chemical inducer of dimerization.
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.
Belshaw
P J
PJ
Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA. sls@slsiris.harvard.edu
Spencer
D M
DM
Crabtree
G R
GR
Schreiber
S L
SL
D000591:Amino Acid Isomerases / Q000378:metabolism*; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016572:Cyclosporine / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D019281:Dimerization; D053222:Fas Ligand Protein; D005470:Fluorometry; D015870:Gene Expression / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D006651:Histocytochemistry; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000494:pharmacology; D008954:Models, Biological; D015394:Molecular Structure; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D019696:Peptidylprolyl Isomerase; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014157:Transcription Factors / Q000037:antagonists & inhibitors; D014162:Transfection / Q000235:genetics
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D016572:Cyclosporine; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase