鑑定梨形鞭毛蟲mitosome相關因子與囊體形成之間的關係

Abstract

早期認為梨形鞭毛蟲 (Giardia lamblia) 不具粒線體，不過近代研究於小胞器膜上發現鐵硫蛋白 (Iron-sulfur cluster, Isc)，說明梨形鞭毛蟲的祖先曾經擁有粒線體，只是在演化過程中逐漸捨棄許多原本應有的功能，發展出具有部分粒線體功能的囊泡，稱為mitosome，我們實驗室先前發現未知功能的骨髓性白血病因子 (Myeloid leukemia factor, MLF) 出現在未知的囊泡，利用IFA (Immunofluorescence assay) 來共染ISCU (mitosome marker) 和MLF的表現，發現兩種囊泡並沒有共定位，確認MLF定位於mitosome以外的囊泡中，而設計突變蛋白與MLF共染IFA則有共定位，可能MLF參與自噬作用和蛋白酶體 (proteasome) 降解，此外觀察到梨形鞭毛蟲在囊體形成過程中，MLF表現增加，推斷其與囊體形成有關；本次實驗透過選殖八種mitosome相關蛋白探究：(1) 八種mitosome相關蛋白在梨形鞭毛蟲滋養體與囊體形成過程時期中的表現，判斷這些蛋白功能是否與囊體形成有關；(2) 八種mitosome相關蛋白在蟲體的定位，以及其與MLF蛋白的位置關係；根據Western blot結果顯示，梨形鞭毛蟲在過度表現7188、8148、9296、10971、12229、16386、16891或22587蛋白的情況下，MLF蛋白表現上升，而過度表現所有八種蛋白的情況下，Bip蛋白表現增加，過度表達蛋白7188、9296、10971或16386讓CWP1 (Cyst wall protein 1) 表現下降，但其餘蛋白的高濃度表現讓CWP1上升，而IFA顯示的各自表達8148、9296、10971、12229、16386、16891或22587的囊泡和MLF所在囊泡有出現部分的共定位。

People used to believe that Giardia lamblia did not have mitochondria. However, recent studies have found iron-sulfur clusters (Isc) on its organelle membranes, suggesting that ancestors of Giardia once possessed mitochondria but gradually lost many of their original functions over the course of evolution, and then evolved vesicles called mitosomes, which retain some mitochondrial functions. Our lab identified an unknown molecule, named Myeloid Leukemia Factor (MLF) appearing in unidentified vesicles. They used Immunofluorescence Assay (IFA) to co-stain ISCU (mitosome marker) and MLF, finding no co-localization between two proteins. However, a mutant protein designed for co-staining with MLF in IFA showed co-localization, confirming that MLF is located in vesicles except mitosome and potentially involved in autophagy and proteasome degradation. In this study, we cloned eight mitosome-related proteins to investigate：(1) their expression in Giardia lamblia during trophozoite and encystation stage, assessing whether these proteins are involved in encystation, (2) their location within the organism and spatial relationship with MLF. According to the Western blot results, Giardia lamblia shows increased MLF expression levels upon overexpressing protein 7188, 8148, 9296, 10971, 12229, 16386, 16891 or 22587. In the case of overexpression of all eight proteins, Bip protein increases. An overexpressed protein 7188, 9296, 10971 or 16386 decreases CWP1, whereas overexpression of the other proteins increased CWP1 expression. The immunofluorescence analysis (IFA) showed partial colocalization between vesicles expressing 7188, 8148, 9296, 10971, 12229, 16386, 16891 or 22587 and the vesicles containing MLF.