陰道滴蟲PI4P5K參與在鐵刺激PIP2產生的功能角色

The role of a Phosphatidylinositol-4-phosphate-5-kinase in iron-inducible PIP2 production of the human pathogen, Trichomonas vaginalis.

林書帆(Shu-Fan Lin)

指導教授 ： 許弘明

國立臺灣大學/醫學院/微生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803376

摘要

前人利用磷酸化蛋白質體學發現陰道滴蟲細胞膜上的磷脂酸肌醇4, 5-雙磷酸(TvPI4P5K)在鐵刺激5分鐘後，其第七號絲胺酸的磷酸化上升4至5倍。在高等真核細胞中PI4P5K主要的功能是將PIP磷酸化形成PIP2，在本研究利用免疫螢光染色及免疫斑點實驗發現PIP2主要分布在陰道滴蟲細胞膜上並且表現量與鐵離子濃度呈正相關。在鐵離子刺激下， PIP2訊號會在15分鐘內達到最高，並在30分鐘後下降至起始濃度，同時，PLC抑制劑處理會造成PIP2持續累積，顯示PIP2可能經由PLC水解後引發下游訊號傳遞。另外，受鐵誘發PIP2上升的現象與控制組相比，在大量表現TvPI4P5K野生型及S7D突變的細胞株中訊號明顯上升，而在K136A及S7A突變的細胞株中訊號則較低。以免疫共沉澱法找到與TvPI4P5K作用的TvARF-1，也共同參與TvPI4P5K主導受鐵誘發PIP2上升的現象。本研究首次在陰道滴蟲發現鐵離子會透過訊息傳導引起TvPI4P5K磷酸化同時與TvARF-1形成複合體進而調控PIP2生成，其分子機制與細胞生物功能仍待進一步釐清。

關鍵字

陰道滴蟲 ； PI4P5K ； 磷脂酸肌醇4, 5-雙磷酸 ； 鐵離子刺激 ； 訊號傳遞

並列摘要

Iron essential to most organisms was found to trigger numerous signaling pathways in a human pathogen, Trichomonas vaginalis. In our preliminary phosphoproteomic analysis, phosphorylation of Ser7 in a phosphatidylinositol-4-phosphate-5-kinase-like protein (TvPI4P5K) was detected to a level higher in cells upon iron repletion. Given that the known function of mammalian PI4P5K is to convert phosphatidylinositol 4-phosphate (PIP) to phosphatidylinositol 4, 5-bisphosphate (PIP2

), our preliminary data strongly suppose that iron-induced signaling may regulate biosynthesis of PIP2 through TvPI4P5K catalysis in this parasite. To validate this, immunofluorescence assay and dot blot analysis detected by anti-PIP2 antibody were exploited. The majority of PIP2 signal was detected on plasma membrane with the intensity related to iron amount in culture medium. Iron was also shown to transiently induce intracellular PIP2 biosynthesis to peak within 15 min of iron repletion, and then decline to the basal level post 30 min of iron repletion. Interestingly, PIP2 level was accumulated when all cells were pretreated with phospholipase C (PLC) inhibitor, suggesting that PIP2 amount may be tightly regulated in this parasite for downstream signaling activation. In the meanwhile, iron-mediated intracellular PIP2 level was respectively accelerated and suppressed in transgenic cells overexpressing wild type and dominant-negative mutants of TvPI4P5K, elucidating the activity of TvPI4P5-K in PIP2 production. When immunoprecipitation coupled with gel-based protein identification with mass spectrometry was explored, an ADP-ribosylation factor 1 (TvARF-1) was found to form the protein complex with TvPI4P5K and associated to TvPI4P5K-mediated PIP2 production. However, more molecular evidence in functional characterization of TvPI4P5K and its interacting counterparts are needed, our findings in PIP2 regulatory mechanism will open a new dimension on the thinking of signal transduction in the protozoan scientific community.

並列關鍵字

Trichomonas vaginalis ； PI4P5K ； PIP2 ； iron-inducible ； signaling pathway

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