陰道滴蟲內 Myb1 與 Cyp A-1 蛋白質之交互作用

Abstract

陰道滴蟲中附著蛋白質 ap65-1 基因之表現受到鐵離子與生長環境改變而影響。前人發現鐵能促進 Myb1之核輸送，以抑制受鐵誘發之 ap65-1 轉錄活性。 前人以大腸桿菌雙雜交系統(bacterioMatch Two-Hybrid System)篩選陰道滴蟲 cDNA 基因庫，得到一與 Myb1相互作用之蛋白質，CypA-1，此 cyclophilin-like protein 為脯氨酸異構脢(peptidyl prolyl cis-trans isomerase，PPIase)蛋白質家族中 cyclophilin (Cyp)一類。本研究以 GST-pull down 分析進一步證實，Myb1與 CypA-1為專一性作用。CypA-1 mRNA 表現量因高鐵處理而提高。利用酵素活性分析得知，CypA-1酵素活性 kcat/Km 為103 M-1S-1；而突變株 R63A 酵素活性 kcat/Km 為6×102 M-1S-1。由螢光免疫分析及西方轉漬法發現，CypA-1及 R63A 皆表現於陰道滴蟲細胞質中，而 R63A 的過度表現會減低 Myb1進入細胞核。經片段刪減及單點突變得知，Myb1與 CypA-1作用區域可能為104EYGPKWNKI112。配合 GST-pull down 分析發現，突變株 YGP105-107AAA 與 Myb1作用力減弱，指出 Y105-P107參與兩者間的作用。進一步將 Myb1及其突變株轉染於陰道滴蟲細胞中，發現 Myb1表現在細胞核中，突變株 P107A 聚集成顆粒於核膜附近，突變株 YGP105-107AAA 則表現於細胞質中。以 Cyclosporin A 處理轉染株 Myb1，發現 Myb1或聚集成顆粒或均勻散布在細胞質中，顯示 CypA-1與 Myb1之作用與 Myb1核輸送功能有關。 本研究證實 Myb1與 CypA-1間的交互作用，助於了解 Myb1蛋白質之生理功能，同時也提供了 Myb1於陰道滴蟲基因調控上ㄧ新的研究方向。

Expression of the adhesion protein ap65-1 gene in the human pathogen, Trichomonas vaginalis, is critically regulated by iron, which was shown to enhance the nuclear translocation of Myb1 that may inhibit iron-activated transcription of the ap65-1 gene. In this study, bacterioMatch Two-Hybrid System (Stratagene) was applied to identify potential Myb1 interaction proteins. One of Myb1 interacting partner proteins was identified, and named CypA-1. CypA-1 is a cyclophilin-like protein, a family of highly conserved ubiquitous proteins in all the organisms. Specific interaction between CypA-1 and Myb1 was confirmed by the GST-pull down assay. CypA-1 mRNA level increased with iron treatment. Recombinant CypA-1 was assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, and gave a value of kcat/Km 103 M-1S-1. R63A retained 60 % of the catalytic efficiency (kcat/Km) of wild-type recombinant CypA-1. Based on the immunofluorescence staining and Western blot assay, both CypA-1 and R63A were expressed in the cytoplasm. Expression of R63A inhibited the nuclear translocation of Myb1. The region of Myb1 interacting with CypA-1 was found to be E104-I112. YGP105-107AAA also interacted with CypA-1 with decreased activity as reveal by the GST-pull down assay. Based on the immunofluorescence staining, Myb1 expressed in the nucleus, P107A was accumulated close to the nuclear membrane, but YGP105-107AAA was evenly distributed in the cytoplasm. Results indicate the participation by Y105-P107 in the translocation of Myb1. Myb1 was either expressed as accumulated close to nuclear membrane, or in the cytoplasm by cyclosporin A treatment. These results provide a new aspect for investigation on the role of the Myb1 protein in transcription regulation of Trichomonas vaginalis.