臺灣博碩士論文加值系統

篦蔴子毒蛋白係屬於第二型核糖體去活性蛋白質家族成員之一，是由兩個單元藉由一對雙硫鍵所組成，包括一個具有酵素活性的A鏈(RTA)以及一個能與半乳糖結合的B鏈(RTB)。篦蔴子毒蛋白最早係自大戟科篦蔴屬植物的種子中萃取而得。目前對於篦蔴子毒蛋白如何進入細胞中及其作用機制為何，科學家們已有了一些的研究與瞭解。首先，篦蔴子毒蛋白藉由其RTB與細胞膜表面上接受器的半乳糖單元結合，並經由內噬作用進入細胞中，經過轉運作用，最後RTA會利用其RNA N-醣苷水解酶活性專一性地移除28S核糖體RNA上第4,324個腺嘌呤殘基，使得核糖體失去正常活性，而達到抑制蛋白質的生合成，進而毒殺細胞。
近年來的研究報告顯示，篦蔴子毒蛋白除了透過抑制蛋白質轉譯作用而造成細胞死亡，亦可透過誘發計劃性程式凋亡的途徑而達成其毒殺細胞的作用。在本實驗室，過去致力於篦蔴子毒蛋白如何誘發計劃性程式凋亡的相關機制之探討，發現BAT3係一個RTA結合蛋白質，並且證實BAT3在篦蔴子毒蛋白誘發細胞凋亡中所扮演的關鍵性角色。此外，BAT3會被篦蔴子毒蛋白所活化的第三型細胞凋亡酶所切割，產生一個羧基端131個胺基酸殘基的片段，命名為CTF131。令人驚奇地，在人類子宮頸癌細胞株(HeLa cells)中表現CTF131片段會造成許多細胞凋亡形態上的特徵，包括磷酯絲胺酸外暴到細胞膜外、細胞形狀圓起和皺縮、細胞核緊緻化、肌動蛋白質遭受破壞等。在本研究中，為了要探索與CTF131誘發細胞凋亡及形態變化有關的分子，酵母菌雙雜合篩選系統被用來尋找細胞內的CTF131結合蛋白質。我們成功地找到了兩個CTF131結合蛋白質，分別鑑定為PLK4和PRDX3。因為CTF131對PLK4有較強的結合能力，所以本研究將著重於探討CTF131與PLK4的交互作用，以及PLK4可能在篦蔴子毒蛋白誘發細胞凋亡中所扮演的角色。經由免疫共沉澱和免疫細胞染色的方法，我們更進一步證實了CTF131與PLK4在細胞中的確具有相互結合的關係。從共軛焦顯微鏡觀察，發現PLK4會在有絲分裂時期均勻地分布在染色體上。藉由試管內激活酶分析，我們也發現了CTF131會抑制PLK4的激活酶活性。總結來說，本研究對於篦蔴子毒蛋白誘發細胞凋亡提供了一個嶄新的觀點與解釋，CTF131藉由抑制PLK4活性而影響紡綞絲的形成與功能，導致有絲分裂進行受阻，進而達到毒殺細胞的作用。

Ricin is a powerful toxin protein and belongs to one of typeⅡ ribosome inactivating proteins (RIPs) family, composing of a toxophoric A-chain (RTA) and a galactose-binding B-chain (RTB). Originally, ricin was isolated from the seeds of Ricinus communis (castor beans). The RTB possesses a lectin activity which can bind to the galactose moiety of glycoproteins or glycolipids on the cell membrane and facilitate the receptor-mediated endocytosis, while the RTA utilizes its RNA N-glycosidase activity to specifically remove the 4,324th adenine residue from 28S rRNA and inhibit protein biosynthesis.
Recently, several evidences revealed that ricin triggered cell death not only through inhibition of protein translation but also through induction of apoptosis by activating caspase-3 activity. In the previous study, it had been demonstrated that BAT3 (human HLA-B-associated transcript 3) was a RTA-binding protein and it was quite crucial for ricin-induced apoptosis. Moreover, BAT3 was able to be cleaved by ricin-induced caspase-3 activity and released a C-terminal 131 residues fragment designated as CTF131. Surprisingly, overexpression of CTF131 in HeLa cell resulted in several characteristics of apoptotic features, including phosphatidylserine (PS) exposure, cell rounding and shrinkage, nuclear condensation, and F-actin disruption. To further understand the precise molecular mechanism about CTF131-induced apoptotic morphological changes, a yeast two-hybrid system using CTF131 as bait was employed to identify CTF131-binding proteins. Two clones were isolated during this screening. Through nucleotide sequencing and database search, these two clones have been shown to encode polo-like kinase4 (PLK4) and peroxiredoxin3 (PRDX3), respectively. Since PLK4 had a stronger binding affinity with CTF131 than PRDX3, the former was chosen as a major target in this study. The in vivo specific interaction between CTF131 and PLK4 was demonstrated by using co-immunoprecipitation and immunostaining. Our observations showed that PLK4 protein localized in the nuclei among chromosomes at M-phase, indicating that both of CTF131 and PLK4 were indeed physiologically relevant in cells. By in vitro kinase assay, it was suggested that CTF131 might inhibit the kinase activity of PLK4, using casein as substrates. Overall, to gain a new insight into ricin-triggered apoptosis, CTF131 perhaps contributed to ricin-induced apoptotic morphological changes by suppression of PLK4, affecting spindle dynamics and mitotic progression.

Abbreviations…………………………………………….…..…………………………….ш
中文摘要……………………………………………………………………………….……Ⅴ
Abstract………………………………………………………………………………..……Ⅶ
Introduction……………………………………..…………………………………………..1
Materials and Methods……………………………………….……………………………7
1.Materials……………………………………………………………………………………..7
2.Plasmid construction………………………………………………………………………...9
3.Plasmid maxi-preparation by ultracentrifugation……..……………………………………15
4.Western blotting analysis…………………………………………………………………...17
5.Yeast two-hybrid system……………………………………………………………………21
6.Cell culture and transient transfection……………………………………………………...28
7.Co-immunoprecipitation…………………………………………………………………....30
8.Immunofluorescence and confocal microscopy…………………………………………….31
9.In vitro kinase assay………………………………………………………………………...33
Results…………….…………………………………………………………………………34
1.Identification of CTF131-interacting proteins by using yeast two-hybrid system…………34
2.Protein expression and intracellular localization of PLK4…………………………………39
3.The specific interaction between CTF131 and PLK4………………………………………41
4.CTF131 could inhibit the kinase activity of PLK4………………………………………....42
Discussion…………………………………………………………...……………...……….43
PLK4 (Sak) gene structure……………………………………………………………………44
Polo/PLK family function…………………………………………………………………….45
The relationship between CTF131 and PLK4………………………………………………..46
Clinical application of immunotoxins………………………………………………………..47
References………………………………………………………...……………….………..66

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