系統識別號 U0026-1408201301464300
論文名稱(中文) 探討SEPT12絲胺酸198磷酸化對於septin絲狀結構之調控
論文名稱(英文) Ser-198 phosphorylation of SEPT12 regulates septin filament fomation
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 101
學期 2
出版年 102
研究生(中文) 施世全
研究生(英文) Shih-Chuan Shih
學號 S16004149
學位類別 碩士
語文別 英文
論文頁數 70頁
口試委員 指導教授-郭保麟
中文關鍵字 SEPT12磷酸化  septin 複合物  spermiogenesis 
英文關鍵字 SEPT12 phosphorylation  septin complex  spermiogenesis 
中文摘要 Septin家族是一群具高度保留性,且有GTP水解酶活性的蛋白。在真核細胞內,SEPTINs會形成絲狀結構,參與調控細胞內的許多生理功能,例如:細胞的移動、細胞型態形成、細胞骨架重整、以及細胞質分裂...等。在結構上,SEPTINs皆具有GTP鍵結功能區以及N、C端的區域。不同的SEPTINs可藉由GTP鍵結功能區之介面(G interface)或N 端與C 端之端點界面(NC interface)彼此結合,形成寡聚體核心聚合物,進一步以尾端與尾端結合的方式形成更高級的結構,例如:絲狀或是環狀結構。Septin聚合物的組合與解離是受到轉譯後修飾進行調控,許多研究指出在酵母菌的細胞周期過程中,septin聚合物的動態變化受到septin磷酸化的調控。然而在哺乳類中,很少有研究指出septin磷酸化是如何對septin的組合及解離進行調控。
SEPT12為睪丸特異性蛋白,在晚期分化的男性生殖細胞中,會形成絲狀或是環狀的結構參與精子型態的分化。在先前研究我們發現SEPT12參與SEPT2-SEPT6-SEPT7-SEPT12-SEPT12-SEPT7-SEPT6-SEPT2(SETP2-6-7-12-12-7-6-2) 寡具體複合物的組合。在臨床上,我們也發現SEPT12的點突變(D197N及T89M)會導致SEPT12無法形成絲狀結構,促使精子的環體缺陷,進而導致男性不孕症。在本研究中,我們藉由質譜儀鑑定SEPT12磷酸化位點,發現只有絲胺酸198(Ser198)受到磷酸化調控。因此我們想要觀察絲胺酸198磷酸化如何調控SEPT12形成絲狀結構。首先,我們將絲胺酸198個別突變成丙胺酸(Ala) 、 天門冬胺酸(Asp)或谷胺酸(Glu)去模擬去磷酸化(S198A)及磷酸化(S198D和S198E)。我們觀察到絲胺酸198在模擬磷酸化的情況下無法與SEPT7結合形成核心結構進而無法形成絲狀結構。藉由轉殖不同量的SEPT12WT及SEPT12S198E,我們發現磷酸化的多寡會影響到SEPT12型成絲狀結構的效率。我們接著去觀察絲狀結構被絲胺酸198磷酸化抗體染到的情形,發現只有少數的絲狀結構有磷酸化絲胺酸訊號。我們更進一步去探討絲胺酸198磷酸化在造精過程中所扮演的角色,在spermiogenesis第14到16步驟,粒線體開始在精子中段部位組裝的過程中,絲胺酸198磷酸化訊號會從頸部的部位移動到環體的部位。總結以上結果,絲胺酸198磷酸化調控對於SEPT12絲狀結構形成及動態移動扮演重要的角色。
英文摘要 Septins, highly conserved P-loop (phosphate binding loop) GTPases and filament-formation proteins in eukaryote, have diverse cellular roles such as migration, cellular morphogenesis, cytoskeletal reorganization, and cytokinesis. All septins contain a consensus GTP binding domain (G interface) and N- and C-terminal region (NC interface). Different septins interact with each other through G and NC interface to form the oligomeric core complexes that join end to end to form the higher-order structures (e.g. filaments, and rings). The assembly and disassembly of septin complexes are regulated by post-translational modifications of component septins. Many studies indicated that the septin phosphoryaltion regulated the septin dynamics during the cell cycle in yeast. However, how septin phosphorylation regulates the assembly/disassembly of septin complexes in the mammalian species is still unclear.
SEPT12 is a testis-specific protein which forms the filament or ring structure in the terminally differentiated male germ cells. In previous studies, we found SEPT12 participates in assembly of the oligomeric core complex-“SEPT2-SEPT6-SEPT7-SEPT12-SEPT12-SEPT7-SEPT6-SEPT2” (SEPT 2-6-7-12-12-7-6-2) to form the filament. Moreover, we found the SEPT12 mutations (D197N and T89M), which lose the ability to form the filament and cause male infertility with defective sperm annulus. In this study, we identified that Ser198 was the only phosphorylation site by using mass spectrometer. Therefore, we aimed to investigate the effects of Ser198 phosphorylation on SEPT12 filament assembly/disassembly. First, Ser198 to Ala or Asp/Glu mutants were generated to mimic dephosphorylation (SEPT12S198A) or phosphorylation (SEPT12S198D and SEPT12S198E), respectively. The SEPT12S198D and SEPT12S198E neither formed filament nor interacted with SEPT7. By co-transfecting different doses of wild-type SEPT12 and the SEPT12S198E plasmids into cells, we found the efficiency of filament formation was decreased with increasing doses of SEPT12S198E. We also observed that the filament was rarely stained by the specific p-Ser198 antibody. We went further to explore the p-Ser198 expression pattern in spermatogenesis. The p-Ser198 signal was localized at the middle piece during the step14-16 of spermiogenesis when mitochondria start to rearrange along middle piece. In summary, our finding suggests Ser198 phosphorylation is important for formation of SEPT12 filament and dynamic of SEPT12 complex.
論文目次 Chinese Abstract(摘 要) I
Abstract III
1.1 Male infertility 1
1.2 Spermatogenesis and spermiogensis 2
1.3 The genes involve in male infertility during the spermatogenesis 4
1.4 Septin gene family 4
1.5 Setpin related diseases 6
1.6 The role of Septin phosphorylaion 7
1.7 Setpin12 9
1.8 Objectives 10
2.1 Site- directed mutagenesis 11
2.2 Cell culture 12
2.3 Total protein extraction 13
2.4 Co-Immunoprecipitation analysis (Co-IP) 14
2.5 The analysis of phosphorylation profile by mass spectrometer 16
2.5.1 Liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis 16
2.5.2 Database search: 17
2.5.3 Phosphorylation site localization: 17
2.6 The generation of Ser198 phosphorylation antibody 17
2.7 Western blot analysis 17
2.8 Sperm preparation 19
2.9 Separation of the testicular germ cell population 19
2.10 Immunofluorescent assay, IFA 21
2.10.1 For testicular section embed in paraffin blocks 21
2.10.2 For sperm smear preparation 22
2.10.3 For cell 24
2.11 His- tagged protein purification 25
2.12 Silver staining 27
2.13 GTP binding assay 28
2.14 Phosphorylation profile prediction 29
2.15 Statistical analysis 29
3.1 Identification of SEPT12 phosphorylation sites by mass spectrometer 30
3.2 The constitutively phosphorylated Ser198 loses the capability to form the filament 30
3.3 The mimetic phosphorylated Ser198 interferes with interaction between SEPT12/SEPT7 and SEPT12/SEPT2 31
3.4 Ser198 phosphorylation disrupts the formation of octameric SEPT2-6-7-12-12-7-6-2 complex 32
3.5 The Ser198 phosphorylation does not affect GTP binding 33
3.6 The SEPT12 filament is hardly stained by the anti p-Ser198 antibody 33
3.7 The expression pattern of phorphorylated Ser198 in terminal post-meiotic male germ cells 34
4.1 The phosphorylation site of SEPT12 35
4.2 How Ser198 phosphorylation affects SEPT12 complex assembly 36
4.3 The potential kinase or phosphatase regulate Ser198 phosphorylation 38
4.4 The possible biological function of Ser198 phosphorylaion 39
4.5 Conclusions 40
References 41
Figures 49
Table 69
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