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標題: | 自噬作用相關藥物對梨形鞭毛蟲壓力相關蛋白質的影響 The Impact of Autophagy-related Drugs on Giardia lamblia Stress-related Proteins |
其他標題: | The Impact of Autophagy-related Drugs on Giardia lamblia Stress-related Proteins |
作者: | 蔡善珍 Shiang-Tian Chua |
指導教授: | 孫錦虹 Chin-Hung Sun |
關鍵字: | 梨形鞭毛蟲,自噬作用,壓力反應,MLF,ATG8, Giardia lamblia,autophagy,stress response,MLF,ATG8, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | Giardiasis為梨形鞭毛蟲造成的寄生蟲感染疾病,為目前最常見的腸道原蟲類寄生蟲疾病,主要傳播媒介為受污染的水源。梨形鞭毛蟲的寄生型態為滋養體,可在腸道裡進行複製生長,一部分滋養體進行囊化形成囊體,隨宿主的糞便排出體外。真核生物在細胞內進行物質降解與回收的途徑有兩種,分別是自噬作用(autophagy)及Ubiquitin-proteasome system(UPS)。雖然有研究團隊在飢餓狀態,或是以metronidazole(MTZ)處理的梨形鞭毛蟲滋養體看到類似autophagy的現象,但我們對梨形鞭毛蟲的自噬作用仍然不甚了解。過去實驗室發現梨形鞭毛蟲在藥物作用下, ATG8、FYVE及MLF的表現量均會上升,而這三種蛋白同時出現的囊泡數量也會增加。因此推測這幾種蛋白質都和梨形鞭毛蟲的自噬作用有關。梨形鞭毛蟲的26S proteasome在滋養體正常生長狀態時會呈點狀散佈在細胞質和細胞核內。RPN11是26S proteasome的subunit,在梨形鞭毛蟲承受壓力時也會跟著上升。WRKY及MBF1乃梨形鞭毛蟲的壓力反應基因調節相關因子。
LY294002可結合在PI3K的ATP-binding位置,並抑制其kinase活性。Temsirolimus 是一種mTOR 抑制劑,其分子在細胞內大部分會轉換成rapamycin,進而促進autophagy作用。NH4Cl會使溶酶體的酸鹼值上升,進而抑制其降解能力,故也會使autophagosome堆積。Methyl methane sulfonate(MMS)會對DNA的鹼基進行烷化,破壞DNA結構,輕者誘使DNA修復機制的發生,重者造成雙股螺旋斷裂和誘發Nonhomologous end join (NHEJ)。Etoposide可以抑制Topoisomerase II並導致DNA double strand break。抗寄生蟲藥物Metronidazole(MTZ)在細胞內會被還原成活化態,攻擊寄生蟲的DNA與蛋白質,進而導致死亡。Artemisinin是有效的抗瘧疾藥物,其endoperoxide bridge會在紅血球內被打破並產生自由基,進而殺死瘧原蟲。Ivermectin是用於治療線蟲與節肢動物引起的感染疾病,也具有透過抑制AKT/mTOR訊息傳導路徑而促進autophagy的作用。Glyphosate是廣泛運用在除草的農藥,動物細胞研究發現此藥物會使細胞產生氧化壓力和促進自噬作用的發生。 MLF、FYVE、BiP及RPN11蛋白質在Temsirolimus、NH4Cl、Etoposide、MTZ、Artemisinin、Ivermectin及Glyphosate作用24小時後皆會增加表現。MMS會使FYVE及RPN11的蛋白質表現量上升。LY294002會使MLF、FYVE及BiP的蛋白質表現量上升。ATG8蛋白質表現量在NH4Cl、MMS、MTZ、Artemisinin作用24小時後呈上升趨勢,但在LY294002的作用下卻下降了。CWP1在NH4Cl、Artemisinin、Ivermectin及Glyphosate的作用 下呈上升趨勢,而在LY249002及Temsirolimus的作用下卻呈下降趨勢。WRKY及MBF1這兩種蛋白質則在大部分的藥物作用之後皆增加表現。另外發現六種未知功能的蛋白質(暫時分別命名為17276、3491、8358、7035、4852、27910)會表現於部分MLF囊泡,而其中17276、3491、8358、7035表現量在囊化過程中呈上升趨勢,4852、27910則呈相反趨勢。與對照組相比,大量表現17276-HA、3491-HA、8358-HA、7035-HA蛋白質的細胞株,其細胞壓力相關蛋白質MLF及BiP、及囊體化相關蛋白質CWP1的表現量也會增加。大量表現4852-HA、27910-HA蛋白質雖然也會使BiP的表現量上升,卻會降低MLF及CWP1蛋白質的表現。 本研究從細胞壓力反應(BiP、RPN11、FYVE、ATG8、MLF)、分化(CWP1)及基因調控(WRKY、MBF1)三個層面證實梨形鞭毛蟲在藥物導致的細胞壓力下會提升這些蛋白質的表現量。而四種未知功能的蛋白質17276、3491、7035及8358可能與梨形鞭毛蟲的壓力反應及囊化作用有關,但扮演何種角色仍有待釐清。 Giardiasis, a waterborne parasitic disease, is one of the most common intestinal protozoa parasite infection in the world. It is caused by the infection of Giardia lamblia, a single cell flagellated protozoa. Giardia lamblia trophozoite lives and replicates in the human intestine. The cysts are discharged into the environment along with host stool and ready to infect their next host. Eukaryotic cells break down and recycle cellular component mainly through two pathways, autophagy and ubiquitin-proteasome system (UPS). Although autophagosome-like structure was observed in starved or metronidazole (MTZ)-treated G. lamblia trophozoites, it is still unclear how autophagy is carried out in this parasite. Our lab found that Giardia expressed more ATG8, FYVE and MLF proteins following certain drugs treatment, as well as vesicles expressing these proteins. We thus speculated that these proteins may involve in Giardia autophagy. Giardia 26S proteasomes scatter throughout the trophozoite in nourished environment. RPN11, a subunit of 26S proteasome, was found to increase when the parasites were stressed, e.g. starved. WRKY and MBF1 are both transcription factors which are related to stress response. In this work, certain drugs were used to treat Giardia lamblia trophozoites in order to induce stress. LY294002 binds to the ATP-binding site of PI3K to inhibit its kinase activity. Temsirolimus is mTOR inhibitor which converts to rapamycin, or acts as itself, in the mammalian cells to induce autophagy. NH4Cl is lysosome inhibitor which causes autophagosome accumulation. Methyl methane sulfonate (MMS) causes DNA methylation and structure destruction, such as double-strand break or non-homologous end joining (NHEJ). Etoposide inhibits Topoisomerase II and causes DNA double-strand breaks. Antiparasitic drug metronidazole (MTZ) is reduced to radical form in the parasitic protozoa and attacks cellular proteins and DNA. Artemisinin, an effective antimalarial drugs, generates a radical unit when its endoperoxide bridge is broken in erythrocytes to kill Plasmodium. Ivermectin is a common drug to treat nematode and insect infectious disease. Glyphosate is a widely used herbicide which is found to induce oxidative stress and autophagy in animal cells. MLF, FYVE, BiP and RPN11 proteins were increased following treatment of temsirolimus, NH4Cl, etoposide, MTZ, artemisinin, ivermectin and glyphosate. FYVE and RPN11 proteins were increased after MMS treatment. MLF, FYVE and BiP were increased by LY294002. ATG8 expression was elevated after NH4Cl, MMS, MTZ and artemisinin treatment but decreased after LY294002 treatment. CWP1 was increased after NH4Cl, artemisinin, ivermectin, and glyphosate treatment, but decreased after LY249002 and temsirolimus treatment. WRKY and MBF1 were increased in most of the experiment group in my work. On the other hand, 6 unknown Giardia proteins (temporarily named 17276, 3491, 8358, 7035, 4852 and 27910) were found co-localized with MLF proteins in certain vesicles of trophozoites cytoplasm. 17276, 3491, 8358 and 7035 protein levels were increased during encystation, whereas 4852 and 27910 acted contrarily. MLF, BiP, and CWP1 were increased as consequence of overexpressing 17276, 3491, 8358 and 7035. Meanwhile, overexpression of 4852 and 27910 caused an increase of BiP but a decrease of MLF and CWP1. My work demonstrates the stress response of G. lamblia following certain drugs treatment in three aspects, i.e. cell stress response (BiP, RPN11, FYVE, ATG8, MLF), cell differentiation (CWP1), and gene regulation (WRKY, MBF1). Meanwhile, four unknown proteins 17276, 3491, 7035 and 8358 may involve in stress response and encystation of G. lamblia, yet their roles are still unclear. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/83300 |
DOI: | 10.6342/NTU202300541 |
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顯示於系所單位: | 微生物學科所 |
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