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In silico and in vitro investigations on the molecular mechanism of steroid hormone response of elephant shark, Callorhinchus milii, progesterone receptor
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| Title: | In silico and in vitro investigations on the molecular mechanism of steroid hormone response of elephant shark, Callorhinchus milii, progesterone receptor |
| Other Titles: | 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 |
| Authors: | 林, 暁智1 Browse this author |
| Authors(alt): | Lin, Xiaozhi1 |
| Keywords: | progesterone receptor | | elephant shark | | cartilaginous fish | | steroid hormone | | mutation | | in silico analysis, MD simulation |
| Issue Date: | 24-Mar-2022 |
| Publisher: | Hokkaido University |
| Abstract: | 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. |
| Conffering University: | 北海道大学 |
| Degree Report Number: | 甲第14834号 |
| Degree Level: | 博士 |
| Degree Discipline: | 生命科学 |
| Examination Committee Members: | (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 |
| Degree Affiliation: | 生命科学院（生命科学専攻） |
| Type: | theses (doctoral) |
| URI: | http://hdl.handle.net/2115/86001 |
| Appears in Collections: | 課程博士 (Doctorate by way of Advanced Course) > 生命科学院(Graduate School of Life Science)
学位論文 (Theses) > 博士 （生命科学）
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