陰道滴蟲Myb2及Myb3轉錄因子受鐵及過氧化氫誘導入核之調控機制

Abstract

陰道滴蟲(Trichomonas vaginalis)為寄生於人類生殖泌尿系統的微小原蟲，引起人類之陰道滴蟲症(trichomoniasis)，是目前世界上最常見的性傳染病病原體之一。陰道滴蟲具有數百個Myb基因，其中已知Myb2及Myb3轉錄因子可共同調控ap65-1致病基因的表現。實驗觀察發現，高濃度的鐵離子可誘使Myb3轉錄因子於短時間內大量輸入並接著輸出細胞核，且Myb2及Myb3均受過氧化氫誘導而產生同樣的現象。本研究主要探討Myb2及Myb3轉錄因子受鐵離子及過氧化氫誘導入核之調控機制，及與Myb3入核及出核功能相關之蛋白結構或位點。激酶抑制劑及活化劑分析結果顯示，鐵離子可能活化陰道滴蟲Gα蛋白，並透過cAMP及cGMP調控下游PKA及PKG活性，同時活化Ras及MEK，影響Myb3的核輸入；而過氧化氫可能透過Ras相關之訊息傳導路徑，調控Myb2及Myb3的核輸入。觀察不同位點突變之Myb3蛋白質受鐵誘導後的入核及出核情形，得知Myb3的核輸入功能至少須要胺基酸序列48-167，且胺基酸156-167附近之數個順式作用因子及磷酸化位置亦與Myb3之入核調控相關。目前針對陰道滴蟲轉錄因子之核輸入機制及訊息傳導路徑了解甚少，本研究可提供初步數據，作為進一步實驗之基礎。

Trichomonas vaginlais that spreads among the humans causes the most widespread nonviral sexually transmitted disease, trichomoniasis. This protozoan parasite harbors numerous Myb transcription factors, among which Myb2 and Myb3 were demonstrated to co-regulate transcription of an iron-inducible virulence gene, ap 65-1. In this study, underlying mechanisms of iron- and hydrogen peroxide-inducible nuclear import of Myb2 and Myb3 were investigated by the inhibitor and activator assays, with results indicating that iron may trigger trimeric G- and Ras- protein- related signaling pathways leading to Myb3 nuclear import, while hydrogen peroxide induces both Myb2 and Myb3 nuclear import via distinct mechanisms. Possible cis-acting and posttranslational modification sites on iron-inducible nuclear translocation of Myb3 were also investigated by depletion mapping and site-directed mutagenesis. The results suggest that the sequence spanning amino acid residues 48-167 is essential for Myb3 nuclear translocation, and inducible nuclear import of Myb3 may be co-regulated by multiple sites. Information generated from this study may provide a preliminary map for further understanding of iron-triggered signal transduction in this important human pathogen.