Date of Award
Doctor of Philosophy
Marc B. Cox
The 52 kDa FK506 binding protein (h52) is a key positive regulator of Androgen Receptor (AR) in cellular and animal models and is an attractive target for the treatment of prostate cancer. Human FKBP52 is a known regulatory protein and co-chaperone that has been shown to play an important role in the regulation of the AR signaling pathway, and in the development of the male sexual phenotype. Cellular studies in mammalian and yeast cells reveal that FKBP52 is a positive regulator of AR, glucocorticoid receptor (GR), and progesterone receptor (PR), potentiating receptor-mediated gene expression up to 60-fold in some systems.
In targeting FKBP52, a thorough investigation and understanding of the structural elements that underlay its function is necessary. This permits a logical approach in targeting specific interaction motifs, such as those that exist between the AR and FKBP52. Here we use a cross-species comparative approach to analyze the mechanisms of potentiation and the functional difference between FKBP52 and Danio rerio (Zebra fish) FKBP52 (DrFKBP52). In this study we have taken advantage of this by comparing their differences to identify additional important domains and residues. Through this study we have identified the FK2 domain, a previously uncharacterized, non-functional domain, as playing a role FKBP52 activity. This observation dispels the notion that the FK1 domain is the sole regulatory domain, specifically the proline-rich loop. Though both have the proline-rich loop, its presence is not indicative of potentiation activity. A genetic selection screen generated in Saccharomyces cerevisiae for gain of potentiation activity, in a library of randomly mutated DrFKBP52 genes, identified two residues: position 111 in the FK1 domain and 157 in the FK2 domain as being the critical residues for activation of receptor potentiation by DrFKBP52. In both the yeast model and mammalian cells, the DrFKBP52 mutation A111V, which is an adjacent residues downstream of the proline-rich loop, confer significant potentiation activity, whereas the same mutation introduced to FKBP52 only slightly elevates activity. Three dimensional crystal structure homology modeling by I-TASSER indicate that when alanine is replaced by valine at position 111 this change affect both the surface charge (to more neutral) and the hydrophobicity (to more hydrophobic) in vicinity. We believe this change induces an open conformation of the proline rich loop notch, allowing for sufficient surface area for AR interaction. A second residue in the FK2 region, T157R, also greatly influences potentiation. Moreover, the DFKBPr52: A111V _T157R double mutant potentiated hormone signaling as well as wild-type hFKBP52. Collectively these results suggest that specific residues in both FK1 and FK2 domain are critical for full activity and are involved in receptor interactions, which potentiates steroid hormone receptor activity. These newly identified domains and residues could possibly become targets for inhibitors as they could be key residues to specifically disrupt AR-FKBP52 association.
Received from ProQuest
DIONDRA CRYSTAL HARRIS
Harris, Diondra Crystal, "A Comparative Approach to Assessing the Functional and Structural Characteristics of Human FKBP52 in the Regulation of Steroid Hormone Receptor Signaling Pathway" (2014). Open Access Theses & Dissertations. 1256.