Progesterone receptor (PR) is a ligand-dependent transcriptional factor which plays important roles in the reproductive biology of vertebrates [1]. A recent study on genome of a cartilaginous fish, elephant shark, has provided an interesting insight into the evolution of gnathostome [2], which makes this fish an ideal model for the comparative analysis and evolutionary study of steroid hormone receptors (SHRs). In the first chapter, I analyzed the amino acid sequence of elephant shark PR which is cloned in our laboratory and compared with PRs of other vertebrates. Results showed that the DNA-binding domain and ligand-binding domain are highly conserved during evolution. The gene expression analysis of synthetic enzymes of steroid hormones indicates the possibility that progesterone, 17OH-progesterone and 5α-dihydroprogesterone is the physiological ligand for elephant shark PR. The ligand responses of full-length and truncated elephant shark PR were investigated and compared with human and zebrafish PRs. The results showed that full-length elephant shark PR can be stimulated by more steroids than human and zebrafish. Elephant shark and human PRs still showed transactivation after the removal of A/B domain while truncated zebrafish PR almost lost the response ability to steroids. Chapter 1 provides an insight into the endocrine system of elephant shark as well as the ligand-dependent transcriptional function of elephant shark PR. In chapter 2, the effects of mifepristone (RU486), a widely used clinical antagonist of human PR, was examined on elephant shark PR. Results showed that RU486 did not inhibit the progesterone-induced activation of elephant shark PR. Gly-722 in human PR corresponding to Cys-528 in elephant shark PR, which is on the helix 3, is considered essential for the antagonistic effects of RU486 [3]. Mutant PRs with cysteine/glycine substitution on helix 3 were constructed to confirm the relevance of this amino acid to the antagonistic effect of RU486 after mutation was recorded. A decline in steroid response of elephant shark PR-Gly528 by 11-deoxycortisol and an increase in activation of human PR-Cys722 were observed. To understand the molecular mechanism of this phenomenon, I investigated the ligand-receptor interactions between PR and 11-deoxycortisol using in silico methods including docking simulation and molecular dynamic (MD) simulation. The simulations provided a possibility that the interaction with cysteine on helix 3 and methionine on helix 7, Met-607 in elephant shark PR corresponding to Met-801 in human PR, is crucial for 11-deoxycortisol binding stability of PR. To confirm the role of the methionine residue, mutant PRs with methionine to glycine mutation on helix 7 were constructed and their steroid-induced activation were tested. The mutant PRs did not show any steroid response, which indicated the loss of interaction between methionine on helix 7 can lead to the loss of steroid hormone response for PR. In order to further understand the actual role of the methionine on helix 7 to PR-11- deoxycortisol interaction, methionine to leucine mutant PRs were constructed and evaluated by in silico analysis. The simulations indicated that methionine to leucine mutation on helix 7 is comparatively stable than methionine to glycine mutation, suggesting that the steroid-induced activation of PRs are maintained after mutation. Chapter 2 provides us with some further understanding into the role of the interaction between ligand and helices in the ligand-binding stability of PR, which has never been described previously.
(主査) 教授 勝 義直, 教授 山下 正兼, 准教授 木村 敦, 准教授 荻原 克益
生命科学院(生命科学専攻)