並列タイトル等In silico 及び in vitro 解析を用いたゾウギンザメのプロゲステロン受容体のホルモン応答性の分子基盤解明
一般注記Progesterone receptor (PR) is a ligand-dependent transcriptional factor which playsimportant roles in the reproductive biology of vertebrates [1]. A recent study on genomeof a cartilaginous fish, elephant shark, has provided an interesting insight into theevolution of gnathostome [2], which makes this fish an ideal model for the comparativeanalysis and evolutionary study of steroid hormone receptors (SHRs).In the first chapter, I analyzed the amino acid sequence of elephant shark PR which iscloned in our laboratory and compared with PRs of other vertebrates. Results showed thatthe DNA-binding domain and ligand-binding domain are highly conserved duringevolution. The gene expression analysis of synthetic enzymes of steroid hormonesindicates the possibility that progesterone, 17OH-progesterone and 5α-dihydroprogesteroneis the physiological ligand for elephant shark PR.The ligand responses of full-length and truncated elephant shark PR were investigatedand compared with human and zebrafish PRs. The results showed that full-lengthelephant shark PR can be stimulated by more steroids than human and zebrafish. Elephantshark and human PRs still showed transactivation after the removal of A/B domain whiletruncated zebrafish PR almost lost the response ability to steroids. Chapter 1 provides aninsight into the endocrine system of elephant shark as well as the ligand-dependenttranscriptional function of elephant shark PR.In chapter 2, the effects of mifepristone (RU486), a widely used clinical antagonist ofhuman PR, was examined on elephant shark PR. Results showed that RU486 did notinhibit the progesterone-induced activation of elephant shark PR. Gly-722 in human PRcorresponding to Cys-528 in elephant shark PR, which is on the helix 3, is consideredessential for the antagonistic effects of RU486 [3]. Mutant PRs with cysteine/glycinesubstitution on helix 3 were constructed to confirm the relevance of this amino acid to theantagonistic effect of RU486 after mutation was recorded. A decline in steroid responseof elephant shark PR-Gly528 by 11-deoxycortisol and an increase in activation of humanPR-Cys722 were observed. To understand the molecular mechanism of this phenomenon,I investigated the ligand-receptor interactions between PR and 11-deoxycortisol using insilico methods including docking simulation and molecular dynamic (MD) simulation.The simulations provided a possibility that the interaction with cysteine on helix 3 andmethionine on helix 7, Met-607 in elephant shark PR corresponding to Met-801 in humanPR, is crucial for 11-deoxycortisol binding stability of PR. To confirm the role of themethionine residue, mutant PRs with methionine to glycine mutation on helix 7 wereconstructed and their steroid-induced activation were tested. The mutant PRs did notshow any steroid response, which indicated the loss of interaction between methionineon 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 andevaluated by in silico analysis. The simulations indicated that methionine to leucinemutation 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 interactionbetween ligand and helices in the ligand-binding stability of PR, which has never beendescribed 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）
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