IMMUNOPHILIN AND RELATED PROTEINS

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Binding proteins for immunosuppressant drugs have been discovered recently in connection with the oligomeric structures of cytoplasmic steroid receptors. The binding proteins form complexes with known immunosuppressant drugs, such as rapamycin, FK5O6 and cyclosporin A and because there are elevations and declines in the functioning of the immune system with aging and other conditions, it seems likely that there exist one or more endogenous immunosuppressant agents whose levels may be under various types of control. In studying the reassembly of the glucocorticoid receptor non-DNA-binding complex form with purified proteins, we discovered a new protein complexed with the FKBP-52 (p55-59) immunophilin. Because this new protein is purified with the FKBP-52 from different cell lines and it appears only when FKBP-52 appears, we conclude that it may function in the signal transduction pathway of immunosuppressant action. Consequently, we plan to pursue the following objectives: (1) Full-length, truncated and mutated FKBP-52 will be overexpressed in the baculovirus system. Site directed mutagenesis will be followed by overexpression of the mutant proteins and all of these proteins will be purified. (2) Overexpressed and purified FKBP-52, hsp90, hsp7O and other cytosolic factors will be reconstituted into high molecular weight complexes, comparable to cellular complexes, and the effects of immunosuppressant drug ligands on these complexes will be assessed. (3) The 59kDa FKBP-52-associated new phosphoprotein will be cloned, overexpressed, purified and characterized. (4) The sequences in FKBP-52 that are responsible for binding to hsp9O, hsp7O, ATP and the 59kDa FKBP-52-associated protein will be determined by mutation of the FKBP-52 cDNA, overexpression of the derivative protein and assembly into the high molecular weight complex. (5) Specific antibodies against FKBP- 52 and its associated phosphoprotein will be developed. These experiments will clarify the signal transduction process involved in the pathway of drug-induced immunosuppression and may contribute to the methods required to discover the endogenous immunosuppressants, results that will directly affect organ and tissue transplantation.