| 摘要: | 陰道毛滴蟲(Trichomonas vaginalis)是一種厭氧性的寄生單細胞原蟲，具有鞭毛，主要寄生於女性的陰道、尿道、子宮，以及男性的前列腺，進而引起滴蟲性陰道炎，此疾病為世界最普遍的直接性接觸感染疾病。目前研究發現，致病的關鍵其中為黏附蛋白質所產生的細胞黏附作用。而基因ap65-1是黏附蛋白質(ap65)家族的一員，分子量為65 道爾吞。調控基因ap65-1的轉錄因子被認為是陰道毛滴蟲內的Myb1與Myb2蛋白質。 在基因ap65-1上有兩段DNA序列可供Myb蛋白質所辨認，分別為MRE1/MRE2r與MRE2f ，進而調控此基因的轉錄機制。研究中發現陰道毛滴蟲內的Myb2蛋白質可以與DNA序列MRE1/MRE2r與MRE2f結合；利用分子生物學的方法截取全長Myb2蛋白質中胺基酸序列從V40到M156的Myb2x截斷蛋白質，且保有與全長Myb2蛋白質相似的DNA結合能力。 在此，利用核磁共振的技術去探討截斷蛋白Myb2x以及分別與DNA序列MRE1/MRE2r與MRE2f結合後在分子動態上的差異性。在14.7Tesla的靜磁場下，藉由使用異核核磁共振脈衝序列量測遲緩速率(relaxation rate)和15N-1H異核交互作用增異參數(NOE)；並利用簡化光譜密度(reduced spectral density mapping)得到光譜密度函數再與結構比較蛋白質的分子動態差異性。未來，將可以繼續探討Myb2x蛋白質更多的詳細結合機制，進而在設計藥物上有所幫助。 Trichomonas vaginalis, an anaerobic, parasitic flagellated protozoan resides mostly in female vagina, urethra, uterus as well as male prostate gland, is the causative agent of the most common nonviral sexually transmitted infections (STIs) in the world, human trichomoniasis. Recently, multiple surface adhesion proteins have been shown to be engaged in Cytoadherence, an essential step, of the T. vaginalis infection. The ap65-1 gene, a member of the adhesion protein 65 (ap65) multigene family, encodes for multiple homologous 65-kDa proteins, was found to be regulated by transcription factors, Myb1 and Myb2 proteins, in T. vaginalis. Two DNA sequences, MRE1/MRE2r and MRE2f, on the gene, ap65-1, is recognized by Myb protein; this discovery deduce the possible involvement of Myb-like transcription factors related to the transcription mechanism within T. vaginalis. From various experiments, evidences show a specific interaction between full-length Myb2 protein with MRE1/MRE2r and MRE2f. We have found that a truncated fragment of Myb2, designated as Myb2x, consisting of amino acid V40–M156 displays similar DNA affinity. This fragment was employed for affinity binding and NMR structure-dynamic studies. By NMR relaxation technology, the difference in dynamics between Myb2x free, Myb2x-MRE1/MRE2r and Myb2x- MRE2f complex forms was resolved. 15N spin relaxation rates and heternuclear (15N-1H) NOE were measured by standard pulse sequences at static magnetic field of 14.7 Tesla. The relaxation data were further analyzed with reduced spectral density mapping approach to deduce the spectral density functions: J(0), J(ωN) and J(ω0.87H) . The protein dynamics of Myb2x, as revealed by the reduced spectral density functions, are mapped onto the structures of the free and DNA-bound forms of Myb2x. The results showed that binding of DNA tighten the protein structure considerably. Such information will be useful for design of effective drugs for the treatment of human trichomoniasis. |
