梨形鞭毛蟲活性氧化物跟壓力相關蛋白質的研究

Abstract

梨形鞭毛蟲(Giardia lamblia)是一種廣泛分布全球的腸內致病性寄生蟲，通常藉由飲用受汙染的水源或食物而感染。梨形鞭毛蟲生活史主要分為兩種，滋養體時期和囊體時期，梨形鞭毛蟲進入宿主後，以滋養體型式生活，在腸道中會進行囊體化成囊體。 自噬作用是生物體內自我降解的作用，調節細胞在生物體內細胞週期的重要機制。在自噬作用中溶酶體(lysosome)會與自噬體(autophagosome)結合，形成自噬溶酶體(autophagolysosome)，AcPh(acid phosphatase)蛋白質是儲存於溶酶體的酵素。蛋白酶體(proteasome)藉由化學反應打斷肽鍵，降解細胞不需要的或受到損傷的蛋白質，RPN11蛋白質是蛋白酶體(proteasome)的蓋子(lid)的成分。BIP蛋白質是chaperone蛋白質，是一個反應ER (endoplasmic reticulum)壓力的指標。ATG8蛋白質是哺乳類動物中的LC3II蛋白質，與自噬作用相關。FYVE蛋白質在人類中發現可促進自噬小體的形成。之前實驗室發現MLF蛋白質會出現在有ATG8和FYVE蛋白質的囊泡中，認為與自噬作用有關。以及在哺乳類動物中Reactive Oxygen Species(ROS)可能導致自噬作用，探討藉由加入藥物，是否會產生ROS的提升。 我們使用AcPh表現滋養體，結果發現BIP、RPN11、ATG8和MLF蛋白質表現量上升；CWP1蛋白表現量皆下降。我們將測試以下對哺乳類動物具有抑制自噬作用且促進化壓力或造成死亡的藥物。細胞處於飢餓狀態，會促進自噬作用。Chloroquine針對溶酶體改變其pH值，使自噬體與溶酶體融合能力下降，導致自噬小體(autophagosome)數目上升。Nocodazole會干擾細胞的微小管(microtubules)，使溶酶體損壞，導致自噬小體(autophagosome)數目上升。MG132是蛋白酶抑制劑，會阻止細胞中的26S蛋白酶作用，使LC3b轉變成LC3b-II使自噬作用上升，誘發細胞凋亡作用。DTT為一種很強的還原劑，藉由影響蛋白的折疊來對內質網產生壓力而促進自噬作用。G418，Geneticin的類似物，阻止蛋白轉譯過程中的延長過程(elongation)，使合成中的多肽鏈合成中止，導致細胞缺乏一些必要蛋白質而死亡。Puromycin使轉譯中的核醣體從肽鏈上脫離，抑制蛋白質轉譯。Curcumin為一種抗發炎藥物，阻斷細胞訊息傳遞，也可以抑制腫瘤細胞增生。Scr7會抑制非同源性末端接合（Non-homologous end joining, NHEJ），目前使用於提升CRISPR-Cas9基因編輯系統的效率。Rapamycin是mTOR抑制劑，藉由抑制mTOR，使LC3b-II表現上升，導致自噬作用上升。H2O2上的未配對電子，會產生氧化壓力，對細胞造成傷害。大腸桿菌( Escherichia coli, E.coli)，測試受到細菌入侵時會產生的自噬作用(Xenophagy)。我們藉由加入螢光物質2′,7′-Dichlorofluorescin diacetate，以測試ROS。實驗結果發現，在梨形鞭毛蟲中進行飢餓實驗，以及加入Chloroquine、Nocodazole、MG132、DTT、G418、Puromycin、Curcumin、Scr7、Rapamycin、H2O2和E.coli後，會使ROS的數值上升。另外有研究發現Cysteine HCl為ROS scavenger，會降低ROS的反應。而培養梨形鞭毛蟲的TYI-33培養液，也含有Cysteine-HCl的成分，因此移除TYI-33培養液的Cysteine-HCl；結果發現移除TYI-33培養液的Cysteine-HCl組別的ROS的數值有上升。 再來我們發現進行飢餓實驗，以及加入Chloroquine、Nocodazole、MG132、DTT、G418、Puromycin、curcumin、Scr7、rapamycin、H2O2和E.coli後，BIP蛋白質表現量有上升，我們認為加入藥物產生ER stress，ER壓力增加使梨形鞭毛蟲中BIP表現量上升。另外發現進行飢餓實驗，以及加入Nocodazole、MG132、DTT、G418、Puromycin、curcumin、Scr7、rapamycin和E.coli後，RPN11蛋白質表現量有上升，我們認為加入藥物會影響梨形鞭毛蟲中26S蛋白酶體的作用。我們發現進行飢餓實驗，以及加入Chloroquine、Nocodazole、MG132、DTT、G418、Puromycin、curcumin、Scr7、rapamycin、H2O2和E.coli後，會使MLF蛋白質表現量上升，我們認為這些藥物導致梨形鞭毛蟲的自噬作用上升。還有加入curcumin、Scr7、rapamycin和H2O2後，結果發現與自噬作用相關的的ATG8、FYVE蛋白質有上升，我們推測這些藥物會使梨形鞭毛蟲的自噬作用上升。另外在AcPh表現滋養體加入MG132、DTT、Scr7、H2O2和E.coli後，結果發現加入MG132、DTT、Scr7、H2O2和E.coli會使梨形鞭毛蟲中AcPh蛋白質表現量上升。另外培養梨形鞭毛蟲的TYI-33培養液，含有Cysteine-HCl的成分，因此移除TYI-33培養液的Cysteine-HCl；結果發現移除TYI-33培養液的Cysteine-HCl組別，在梨形鞭毛蟲中ROS有增加，以及BIP、RPN11、ATG8蛋白質表現量有增加。 從我的發現瞭解了梨形鞭毛蟲的AcPh、RPN11、BIP、MLF、ATG8和FYVE蛋白質在加入自噬作用相關藥物處理後會增加表現。還有加入藥物後，梨形鞭毛蟲中的ROS反應有上升。

Giardia lamblia is a widely distributed intestinal protozoan parasite. People ususally get infected by drinking contaminated water or having contaminated fruits. There are two stages of life cycle in G. lamblia, trophozoites and cysts. When G. lamblia enters hosts, it lives in the trophzoite form, and encysts to form an infectious cyst in intestine. Autophagy is a self-degradative process and an important mechanism in regulation of cell cycle in organisms. During autophagy, lysosomes will combine with autophagosomes to be the autophagolysosomes. AcPh (acid phosphatase) is an enzyme stored in lysosomes, and it is also stored in autophagosomes. Proteasome breaks peptide bonds by chemical reaction to degrade unnecessary or damaged proteins. RPN11 is a component of lid in proteasome. BIP protein is a chaperone protein and marker of ER (endoplasmic reticulum) stress. ATG8 protein is named LC3II protein in mammals, and is related to autophagy. FYVE protein promotes formation of autophagosomes in human. Our lab found that MLF protein located in the vesicles that ATG8 and FYVE protein located in, and we suggested that MLF protein is related to autophagy. Because reactive oxygen species (ROS) will cause autophagy in mammals, we tried to detect ROS level by adding drugs. We analyzed AcPh expression trophozoites, and we observed increased BIP, RPN11, ATG8 and MLF protein expression and reduced CWP1 protein expression. We further added autophagy inhibiting drugs or oxidative-stress promoting drugs and growth-inhibiting drugs in mammals above. Chloroquine inhibits the fusion of autophagosomes with lysosomes by affecting pH value in lysosomes, and causes increased autophagosome formation. Nocodazole interferes microtubules in cells and causes lysosomal damage, resulting in an increase in autophagosome formation. MG132 is a proteasome inhibitor that interrupts the function of 26S proteasome. MG132 makes LC3b transform to LC3b-II and promotes autophagy and induces cell apoptosis. DTT is a strong reducing agent, it causes ER stress by interfering the folding proteins and promotes autophagy. G418 is an analog of Geneticin. G418 interrupts the elongation step in protein translation and blocks polypeptide synthesis and. G418 causes cell death because of lack of necessary proteins. Puromcyin causes premature chain termination during translation to inhibit protein translation. Curcumin is an anti-inflammatory drugs. It disrupts signal transfer and inhibits tumor cell proliferation. Scr7 is an inhibitor of non-homologous end joining (NHEJ) and enhances the efficiency of CRISPR-Cas9 genome editing. Rapamycin, an inhibitor of mTOR, makes increased LC3b-II protein expression and causes autophagy. The unpaired electron of H2O2 causes oxidative stress to damage cells. E.coli can induce autophagy in mammal cells, and can be used to test xenphagy in Giardia. We measured ROS level and used 2′,7′-Dichlorofluorescin diacetate. We found that ROS level elevated after starvation, Chloroquine, Nocodazole, MG132, DTT, G418, Puromycin, curcumin, Scr7, rapamycin, H2O2 and E.coli treatment. A research suggested that Cysteine-HCl is a ROS scavenger and reduces ROS. The TYI-33 medium we cultured G. lamblia also has Cysteine-HCl. We removed Cysteine-HCl of TYI-33 medium, and we found that ROS levels increased in the TYI-33 medium without Cysteine-HCl. Next we found increased BIP protein expression by starvation, Nocodazole, MG132, DTT, G418, Puromycin, curcumin, Scr7, rapamycin, H2O2 and E.coli treatment. These drugs cause oxidative stress in G. lamblia. We suggested that oxidative stress can increase BIP protein expression in G. lamblia. We found increased RPN11 protein expression by starvation, Nocodazole, MG132, DTT, G418, Puromycin, curcumin, Scr7, rapamycin and E.coli treatment. We suggested that these drugs will influence the function of 26S proteasome. We also found increased MLF protein expresson by curcumin, Scr7, rapamycin and H2O2 treatment. We suggested that these drugs promote autophagy in G. lamblia. We found increased ATG8 and FYVE protein expression by curcumin, Scr7, rapamycin and H2O2 treatment. We suggested that these drugs cause autophagy in G. lamblia. We also tested AcPh expression trophozoites by MG132, DTT, Scr7, H2O2 and E.coli treatment. The expression of AcPh is increased after MG132, DTT, Scr7, H2O2 and E.coli treatment. The TYI-33 medium we cultured G. lamblia has Cysteine-HCl. We removed Cysteine-HCl of TYI-33 medium, and we found that ROS increases and increased BIP, RPN11, ATG8 proteins expression after removal of Cysteine-HCl. Our data provide evidence that AcPh、RPN11、BIP、MLF、ATG8 and FYVE protein expression increased after autophagy inhibitor treatment. We also found that ROS level increased after autophagy inhibitor treatment.