鑑定與探討梨形鞭毛蟲Maf-like轉錄因子之特性

梨形鞭毛蟲是一種廣泛存在全球的致病性原蟲。梨形鞭毛蟲生活史中有一段細胞分化的過程稱之為囊體化，此時是滋養體轉變為囊體的過程。屬於AP-1蛋白家族的Maf可以調控許多物種的細胞分化。我們在梨形鞭毛蟲中找到一個近似Maf的蛋白質，經胺基酸序列分析發現蛋白質C端部分具有規律出現的leucine，符合Maf zip的一大特徵，且在這些Leucine前端具有一段胺基酸序列與人類Maf進行DNA binding的序列相似，於是我們將其命名為G. lamblia Maf-like protein 1(glMafl1)，並建立過量表現glMafl1之細胞株pPglMafl1。實驗結果發現此細胞株的囊體壁蛋白CWP1也大量增加。為了探究glMafl1的功能，我們將其DNA binding序列與zip序列設計突變，建立突變株pPglMafl1-m1與pPglMafl1-m2。我們發現大量表現glMafl1時，CWP1-3及調控囊體化之轉錄因子Myb2蛋白質及RNA表現量都會上升，促進生成囊體，然而突變株的CWP1-3、Myb2表現及囊體生成數目則減少。透過免疫沉澱法以及GST融合蛋白質沉澱法我們發現glMafl1會和其他調控囊體化之轉錄因子，如：Pax1、WRKY、PDCD5、Myb2直接結合。因此我們認為glMafl1可能參與一個許多轉錄因子結合形成的複合體，共同調控囊體化相關基因以促進囊體化進行。

關鍵字

梨形鞭毛蟲； Maf ；囊體化；囊體壁蛋白CWP1-3 ； Pax1 ； PDCD5 ； Myb2 ； WRKY

並列摘要

Giardia lamblia is one kind of worldwide protozoan pathogens. There is a cell differntiation stage in Giardia life cycle, named encystation, which is the transformation between trophozoite and cyst. Maf, a member of AP-1 family, can regulate cell differntiation in many spieces.. We found a G. lamblia protein similar to well studied mammalian Maf. By amino acids analyzing, we observed that there were several leucines regularly existed at the C terminal of this G. lamblia protein, just like the higher evolution organisms’ Maf Zip domain. Moreover, there are some amino acids upstream of those regular leucines similar to the human Maf DNA binding site basic residues. Based on these amino acid analyzing, we named this protein G. lamblia Maf-like protein 1 (glMafl1), and constructed the Giardia cell strain, pPglMafl1, to overexpress it. The data showed that the cyst wall protein 1 (CWP1) expressing were also up-regulated in pPglMafl1. To understand the function of glMafl1, we mutated the putative basic DNA binding site and leucine zipper of the glMafl1 leading to mutant cell strains of pPglMafl1-m1 and pPglMafl1-m2. We observed that the cyst wall protein 1-3 (CWP1-3) and encystation regulating-Myb2 gene was up-regulated in pPglMafl1 at protein and RNA level. Overexpressing glMafl1 could induce cyst forming. Moreover, the CWP1-3, Myb2 expression and cyst forming decreased in two mutants. Using immunoprerecipitation assays and GST pull down assays, we found that glMafl1 directly interacts with other encystation relating transcription factors, Pax1, WRKY, PDCD5 and Myb2. We thus suggest that glMafl1 participates in a transcription factor complex, which plays a role in the induction of encystation relating genes expression, and thus promoting encystation of G. lamblia.

並列關鍵字

Giardia lamblia ； Maf ； encystation ； CWP1-3 ； Pax1 ； PDCD5 ； WRKY ； Myb2

參考文獻

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鑑定與探討梨形鞭毛蟲Maf-like轉錄因子之特性

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