鑑定梨形鞭毛蟲的細胞循環激酶同源蛋白質對於囊壁蛋白基因轉錄調控之影響

Abstract

梨形鞭毛蟲是一種腸道寄生性原蟲，它是全世界造成腹瀉最常見的原因之一。梨形鞭毛蟲的生活史分為滋養體以及囊體兩個時期，囊體可以抵抗外界環境，在人體外存活很長的時間。當梨形鞭毛蟲分化成囊體時，蟲體會形成囊壁，cyst wall protein (CWP)是組成囊壁的蛋白，囊體化過程中，cwp基因會大量表現，目前已知的有cwp1，cwp2和cwp3。在人類的研究中發現，cyclin-dependent kinase (Cdk)調控細胞週期以及細胞分化，梨形鞭毛蟲的囊體化過程亦包含細胞週期的改變以及細胞的分化，所以在本篇研究中，我們想要尋找梨形鞭毛蟲中是否有Cdk同源蛋白質，並且想要了解Cdk是否參與在囊體化過程中。 我們在梨形鞭毛蟲資料庫中找到兩個相似於Cdk的蛋白質，Cdk1和Cdk2。它們都具有PSTAIRE-like motif。胺基酸序列的分析顯示梨形鞭毛蟲的Cdk2較相似於人類的Cdk2。我們發現Cdk2的mRNA和蛋白質表現量在囊體化時增加，利用免疫螢光染色，我們發現Cdk2表現在蟲體的細胞質中。其次，在梨形鞭毛蟲中大量表現Cdk2會造成CWP1和Myb2蛋白質表現量上升，也造成cwp1，cwp2，myb2基因的mRNA表現量上升。利用流式細胞儀分析發現，大量表現Cdk2造成G2 phase的細胞增加，顯示蟲體傾向往囊體發展。利用IP kinase assay，我們也發現Cdk2可以磷酸化Myb2蛋白，且磷酸化的情形在囊體化時上升。我們進一步發現磷酸化是位於Myb2中不具DNA結合能力的N端，並且可能的磷酸化位點是位於S/TPXK/R以及S/TPXX序列上的serine和threonine。我們也發現一種Cdk抑制物purvalanol A可以抑制Cdk2的kinase活性，並且加入purvalanol A會造成CWP1和Myb2蛋白質表現量下降，以及cwp1，cwp2，myb2基因的mRNA表現量下降，以及降低囊體的產生。 我們將Cdk2中可能會與cyclin結合的PSTAIRE-like motif以及可能參與ATP結合的148位置的aspartic acid進行突變，並且也刪除kinase domain的一長段胺基酸序列。我們發現相較於wild-type Cdk2，大量表現這三個突變體造成CWP1和Myb2蛋白質表現量下降，cwp1，cwp2，myb2基因的mRNA表現量也下降。並且也造成G2 phase的細胞數下降，以及kinase的活性降低。本研究中我們發現Cdk2會磷酸化Myb2蛋白，顯示Cdk2可能參與在囊體化過程中。

Giardia lamblia is a flagellated intestinal protozoan parasite. It is one of the common causes of diarrheal disease throughout the world. The life cycle of G. lamblia can be divided into trophozoite and cyst stages. The cyst can resist to environment and survive outside of human host for a very long period. When G. lamblia differentiates into cyst, cyst wall will form. Cyst wall protein (CWP) is the protein component of cyst wall. During encystation, cwp genes are highly up-regulated. The known cwp genes are cwp1, cwp2, and cwp3. In human studies, cyclin-dependent kinase (Cdk) regulates cell cycle and cell differentiation. Giardia encystation also includes cell cycle progression and cell differentiation. In this study, we want to find whether there is Cdk homologues in G. lamblia and to determine whether Cdk is involved in Giardia encystation. We found two proteins similar to Cdk in G. lamblia genome database, termed Cdk1 and Cdk2. Both of them contain PSTAIRE-like motifs. Amino acid sequence analysis showed that giardial Cdk2 is similar to human Cdk2. We found mRNA and protein levels of Cdk2 increase during encystation. Using Immunofluorescence assay, we found Cdk2 localize in the cytoplasm of G. lamblia. Overexpression of Cdk2 causes an increase of CWP1 and Myb2 protein, and cwp1, cwp2, and myb2 mRNA. Using flow cytometry analysis, we found Cdk2 overexpression increase G2 phase cells. This indicates the differentiation toward cyst. Using IP kinase assay, we found that Cdk2 can phosphorylate Myb2 protein. We further discovered that the phosphorylation of Myb2 is at its N-terminal region which does not have DNA-binding ability. We also found the possible phosphorylation sites are serines and threonines within the S/TPXK/R and S/TPXX motifs. We also found a Cdk inhibitor purvalanol A can inhibit Cdk2 kinase activity. Addition of purvalanol A decreases CWP1 and Myb2 protein, and cwp1, cwp2, and myb2 mRNA, and also decreases cyst production. We mutated Cdk2 at the PSTAIRE-like motif that may be involved in cyclin-binding and the aspartic acid at residue 148 that may be involved in ATP-binding. We also deleted a large portion of sequences within its kinase domain. We found compared to wild-type Cdk2, overexpression of Cdk mutants decrease CWP1 and Myb2 protein, and cwp1, cwp2, and myb2 mRNA expression. Compared to wild-type Cdk2, the mutants also cause decreases of G2 phase cells and kinase activity. In this study, we found that Cdk2 can phosphorylate Myb2 protein. Cdk2 may be involved in encystation in G. lamblia.