鑑定梨形鞭毛蟲的一個新的Helix-Turn-Helix蛋白質

Abstract

梨形鞭毛蟲是一種腸道內原蟲寄生蟲，轉變為感染型囊體時，囊壁蛋白質(cyst wall protein, Cwp)的表現量會提高，以形成外層的囊壁，使梨形鞭毛蟲可以生存在宿主體外。Helix-turn-helix (HTH) domain是一種DNA binding domain，在原核真核生物都有發現具有此類domain的蛋白質，在不同生物中扮演調節細胞分化之轉錄調控因子的角色。我們在梨形鞭毛蟲的基因組資料庫中找到一個在C端含有HTH domain的蛋白質，將此HTH蛋白質命名為HTH1。HTH1的胺基酸序列在BLAST搜尋比對後發現相似於許多真核生物的multiprotein bridging factor1 (MBF1)。人類或果蠅等物種的MBF1都會結合轉錄活化子以活化基因的轉錄，MBF1在許多物種中可促進細胞增生與細胞分化，某些物種的MBF1在死亡時表現量增加。我們發現梨形鞭毛蟲的內生性HTH1基因表現量在mRNA與蛋白質層級上，均為囊體化時期高於滋養體時期，我們將HA-tag接在HTH1並轉染到梨形鞭毛蟲，看到外源性的HTH1也是一樣的趨勢。透過免疫螢光染色我們也發現在滋養體時期及囊體化時期，HTH1均同時表現於梨形鞭毛蟲的細胞核及細胞質中。從電泳位移分析實驗中得知HTH1具有與3種囊壁蛋白質cwp1、cwp2、cwp3基因啟動子結合的能力。在梨形鞭毛蟲大量表現HTH1時會促進Cwp1蛋白質以及cwp1~cwp3基因及其轉錄調控因子之基因myb的mRNA表現量，囊體形成數目也有上升。因此我們推測HTH1可能為參與囊體化相關基因轉錄調控的轉錄因子，並促進囊體的形成。但是若將序列上的HTH domain做不同區段的改變形成突變體則會導致此現象消失；而且當HTH domain內的第1或第2個α-helix突變後，與cwp1基因啟動子結合的能力會明顯下降，推測第1或第2個α-helix的完整性對於HTH1結合DNA的功能有很重要的關係。除此之外我們也發現HTH1和囊壁蛋白質的轉錄因子E2F1、Pax2、和WRKY結合。另外我們加入會造成梨形鞭毛蟲死亡的藥物，包括metronidazole、etoposide及curcumin時，發現HTH1的表現量皆有上升的趨勢，因此HTH1可能與細胞死亡有關。由於HTH1會調控梨形鞭毛蟲的細胞分化，在細胞死亡時表現量上升，又會與轉錄調控因子結合，我們認為梨形鞭毛蟲HTH1與MBF1可能扮演相類似的角色。

Giardia lamblia is an intestinal protozoan parasite. When differentiating into infectious cyst, expression of cyst wall proteins (Cwp) is upregulated. These proteins form cyst wall which encapsulates G. lamblia to help the parasite to survive outside the host. Helix-turn-helix (HTH) domain is a DNA binding domain found in proteins regulating transcription of prokaryotic and eukaryotic creatures. We found a protein with an HTH domain in the C-terminus from G. lamblia genome database and denominated it HTH1. We used BLAST to search proteins with amino acid sequences similar to those of HTH1 and found that HTH1 is similar to multiprotein bridging factor1 (MBF1) from some eukaryotic species. MBF1 in species like human and fruit fly interacts with transcriptional activators to activate transcription. MBF1 promotes cell proliferation and differentiation in different species. MBF1 is upregulated during programmed cell death in some species. We found that both mRNA and protein expression levels of endogenous HTH1 gene during 24 hour encystation were higher than that during vegetative growth stage in G. lamblia. We transfected a construct expressing HA-tagged HTH1 into wild type G. lamblia. Western blot showed that HA-tagged HTH1 was expressed at similar levels during vegetative growth and during 24 hour encystation. Immunofluorescence assay revealed that the HA-tagged HTH1 was localized to the whole cell, including nuclei and cytosol, and expressed at higher protein levels during vegetative growth and encystation. Using electrophoretic mobility shift assay, we found that HTH1 binds to the promoters of cwp1, cwp2 and cwp3 genes which are important genes during encystation. Overexpressing HTH1 in G. lamblia resulted in an increase of levels of Cwp1 protein, mRNA of cwp1~cwp3 genes and a gene of their transcription factor myb, and cyst formation. Therefore we suggest that HTH1 might be involved in transcriptional activation of encystation related genes and promote cyst formation. Using mutated plasmids and transfection assays, we found that the levels of Cwp1 protein, cwp1 and cwp2 mRNA and cyst formation in the HTH1-mutant overexpressing cell line decreased significantly relative to the levels in the wild type HTH1 overexpressing cell line. Additionally, HTH1 with mutation of the first or second α-helix in the HTH domain showed a decreased binding activity to cwp1 promoter. Integrity of these 2 helices is considered important to the DNA binding activity of HTH1. Besides, we found that HTH1 binds to the transcription factors E2F1, Pax2 and WRKY in G. lamblia which regulate the expression of cyst wall proteins. While we treated G. lamblia with lethal compounds, including metronidazole, etoposide and curcumin, HTH1 expression increased. Since HTH1 regulates cell differentiation, has a higher expression level during cell death, and binds to transcription factors; we suggest that G. lamblia HTH1 might play a role similar to MBF1.