The glucocorticoid steroids regulate a wide variety of functions in the body, and their synthetic analogs are used in the therapy of many diseases. In the hormone-free cell, the glucocorticoid receptor, as well as the receptors for mineralocorticoid and sex hormones, exist in a heteroprotein complex that contains three heat- shock proteins - hsp90, hsp70 and hsp56. Dr. Pratt has shown that these three heat- shock proteins bind to each other independent of the presence of steroid receptors, and he postulates that the complex is involved in the very essential cellular functions of protein folding and trafficking. The GR must be bound to this heterocomplex in order for it to bind steroid, and to understand the mechanism of glucocorticoid action, he proposes to study how the GR heat-shock protein complex is formed. Recently, Dr. Pratt has shown that the GR heterocomplex can be formed in an ATP-dependent, monovalent cation-dependent manner by incubating immunopurified GR with rabbit reticulocyte lysate. This enzymatic system apparently unfolds the hormone-binding domain (HBD) of the receptor while converting it into the high affinity steroid-binding conformation. Hsp70 is known to have a protein unfolding activity and the Specific Aim 1 focuses on determining if the protein unfoldase activity of HSP70 is required for forming the GR-hsp90 complex.
Specific Aims 2 and 3 exploit Dr. Pratt's experience with GR heterocomplex formation by lysate fractions to purify and characterize the heterocomplex assembly mechanism, and to determine the order of protein addition in the assembly sequence. One of the proteins in the GR heterocomplex, HSP56, has recently been shown to be an immunophilin that binds FK506, the most potent immunosuppressant drug used to prevent transplant rejection. It is of intrinsic interest that the two drugs that are often used together in immunosuppressive therapy - steroids and FK506 - have binding proteins associating with each other in the same heterocomplex.
Specific Aim 4 explores the relationship between FK506-binding capacity and other components of the complex (i.e., GR and HSP90).
Aim 5 focuses on the assembly of the heat shock protein complex from purified components and the effects of molybdate and endogenous metal factor on the stability of the complex, independent of the presence of the receptor. For several years, Dr. Pratt's laboratory has studied the redox regulation of SH groups in the steroid binding domain as a strict determinant of steroid- binding activity. A vicinal pair of thiols in a subregion of the GR HBD, containing part of both the steroid binding pocket and the HSP90-binding site, has been investigated as a potential site for specific derivatization of the HBD. The goal in Specific Aim 6 is to synthesize an 125I-labeled compound that will derivative this vicinal SH pair and then to use it as a probe for conformational change occurring in the HBD with steroid binding, HSP90 dissociation and heterocomplex reconstitution.
| Pratt, W B; Morishima, Y; Murphy, M et al. (2006) Chaperoning of glucocorticoid receptors. Handb Exp Pharmacol :111-38 |
| Kovacs, Jeffrey J; Murphy, Patrick J M; Gaillard, Stephanie et al. (2005) HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol Cell 18:601-7 |
| Morishima, Yoshihiro; Peng, Hwei-Ming; Lin, Hsia-lien et al. (2005) Regulation of cytochrome P450 2E1 by heat shock protein 90-dependent stabilization and CHIP-dependent proteasomal degradation. Biochemistry 44:16333-40 |
| Peng, Hwei-Ming; Morishima, Yoshihiro; Jenkins, Gary J et al. (2004) Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase. J Biol Chem 279:52970-7 |
| Billecke, Scott S; Draganov, Dragomir I; Morishima, Yoshihiro et al. (2004) The role of hsp90 in heme-dependent activation of apo-neuronal nitric-oxide synthase. J Biol Chem 279:30252-8 |
| Pratt, William B; Galigniana, Mario D; Morishima, Yoshihiro et al. (2004) Role of molecular chaperones in steroid receptor action. Essays Biochem 40:41-58 |
| Pratt, William B; Galigniana, Mario D; Harrell, Jennifer M et al. (2004) Role of hsp90 and the hsp90-binding immunophilins in signalling protein movement. Cell Signal 16:857-72 |
| Harrell, Jennifer M; Murphy, Patrick J M; Morishima, Yoshihiro et al. (2004) Evidence for glucocorticoid receptor transport on microtubules by dynein. J Biol Chem 279:54647-54 |
| Kanelakis, Kimon C; Pratt, William B (2003) Regulation of glucocorticoid receptor ligand-binding activity by the hsp90/hsp70-based chaperone machinery. Methods Enzymol 364:159-73 |
| Pratt, William B; Toft, David O (2003) Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp Biol Med (Maywood) 228:111-33 |
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