陰道滴蟲Myb轉錄因子的入核運輸機制探討

Nuclear localization of Myb transcription factors in parasitic protozoan Trichomonas vaginalis

10.6342/NTU.2013.00901

朱建勳

陰道滴蟲 ； 轉錄因子 ； 脯氨酸異構酶核運送 ； 入核機制 ； Trichomonas vaginalis ； Myb ； cyclophilin ； nuclear localization ； NLS

臺灣大學微生物學研究所學位論文

2013年

博士

戴榮湘

繁體中文

陰道滴蟲細胞中轉錄因子Myb1及Myb2皆可結合鐵誘導基因ap65-1之啟動子調控其表現。本研究發現Myb2之入核控制是透過其結構正確性，以及Myb1之入核受其交互作用酵素TvCyPA1的影響。於陰道滴蟲細胞中過表現HA-fused Myb2或Myb1，利用IFA及western blotting可發現Myb2或Myb1在細胞中皆分布於細胞核。而突變Myb2序列內類似cNLS的鹼性胺基酸無法完全破壞入核能力，顯示其入核機制可能不同一般真核細胞，Myb2中具入核能力之序列為R2R3內胺基酸48-143區域，且若刪除此區域內helix 2~5或RC3任一皆會使Myb2無法進核；若突變helix 2中Isoleucine 74為proline而破壞其二級結構亦無法進核，顯示Myb2結構之完整性可能為入核所需。與Myb2結構組成類似的Myb1則發現相互作用之TvCyPA1蛋白質，可能為脯氨酸異構酵素(peptidyl prolyl cis-trans isomerase)，於陰道滴蟲細胞中過表現HA-CyPA1會促進Myb1入核，過表現HA-CyPA1-R63A突變蛋白會抑制Myb1入核。若突變Myb1之106GP107 dipeptide會破壞與TvCyPA1的結合，其中G106A突變會破壞入核能力但P107A突變卻增加入核比例，可能G106A使TvCyPA1無法結合Myb1導致無法進核，P107A使Myb1留在細胞質的限制消失而增加進核，Myb1之入核可能透過TvCyPA1轉換構型以調控。

In Trichomonas vaginalis, Myb proteins were previously shown to regulate transcription of an iron-inducible ap65-1 gene. In this research, a structure-related nuclear import signal of Myb2 transcription factor was studied, and a cyclophilin A-like protein, referred to as TvCyPA1, was identified to be a binding partner and nuclear localization regulator of Myb1. The HA-tagged Myb2 or Myb1 in T. vaginalis was localized to the nucleus as punctate signals. Myb2 was still localized to the nucleus with mutations in cNLS-like polybasic sequences, suggesting that cNLS may not function in T. vaginali. In Myb2, the sequence spanning amino acid residues 48 to 143, which is embedded within the R2R3 domains was essential and sufficient for protein nuclear import. Myb2 nuclear import was perturbed with point mutation of a conserved isoleucine (I74) in helix 2 to proline that slightly altered secondary structure and ternary folding of the R2 domain, suggesting that nuclear translocation of Myb2 requires a highly ordered structure. Myb1, which has a structure similar to Myb2 was found to interact with TvCyPA1, a cyclophilin A-like peptidyl-prolyl isomerase. Nuclear translocation of Myb1 was respectively enhanced or repressed by overexpressed HA-TvCyPA1 or HA-TvCyPA1-R63A, a catalytic mutant. Point mutation of G106 or P107 in the TvCyPA1-binding motif of Myb1 was demonstrated to disrupt its binding to TvCyPA1. Mutation of G106 in HA-tagged Myb1 resulted in cytoplasmic retention of the protein, while mutation of P107 lead to elevated nuclear translocation, but without affecting its repressive activity. These results suggest that TvCyPA1 may facilitate inter-conversion of the peptidylprolyl bond in the 106GP107 dipeptide to regulate the nuclear importation.