陰道滴蟲中肌動加帽蛋白之功能區域分析

The functional domain analysis for an F-actin capping protein of Trichomonas vaginalis

張景翔(Jing-Yang Chang)

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(2021年)

https://doi.org/10.6342/NTU202103559

摘要

陰道滴蟲感染引起的陰道滴蟲症是最常見的非病毒源性傳染疾病之一，與上皮細胞貼附是其感染宿主的關鍵。在高等真核細胞中，肌動蛋白 (α-Actin) 主導的細胞骨架重組，可以調控細胞型態的轉變，例如利用偽足來進行胞吞作用、貼附爬行等等，而這些相關機制在陰道滴蟲中尚未被深入的探討。根據實驗室前人的發現，陰道滴蟲細胞中肌動加帽蛋白 (F-actin capping protein, TvFACP) 可能藉由抑制肌動蛋白聚合成束，進而造成陰道滴蟲細胞轉型與貼附能力受阻。在本研究中，目標再進一步分析TvFACP蛋白上潛在的肌動蛋白結合觸手 (tentacle) 與酪蛋白激酶 (Casein kinase II) 磷酸化調控位點功能。研究結果顯示，TvFACP蛋白質C端 (237-261胺基酸) 區域刪除，在免疫沉澱實驗中大幅降低與肌動蛋白的結合；若將此蛋白過量表現於陰道滴蟲中，則會降低抑制肌動蛋白重組的能力。而TvFACP蛋白質Ser2與Ser4進行點突變後，在免疫沉澱實驗中仍與肌動蛋白維持高度的結合；將此蛋白過量表現於陰道滴蟲時，卻同樣降低抑制肌動蛋白的重組能力。後續利用固相結合酵素鏈結免疫吸附與共沉降試驗分析，推測TvFACP可同時結合F-actin與G-actin共同調控F-actin聚合。最後以細胞遷移試驗，發現TvFACP過量表現使陰道滴蟲移動能力上升。表示TvFACP藉由結合α-Actin調控細胞骨架重組，影響陰道滴蟲變形與貼附能力。

關鍵字

陰道滴蟲 ； 肌動蛋白 ； 肌動加帽蛋白 ； 肌動蛋白結合觸手 ； 酪蛋白激酶磷酸化調控

並列摘要

Trichomonas vaginalis is the non-viral and sexually transmitted human parasite worldwide. It is the causative agent of trichomoniasis, conjugated to the adverse urogenital disorders. In high eukaryotes, F-actin capping protein (TvFACP) binds on the barbed end of α-actin filament (F-actin) to shape mediate cytoskeletal-based cell morphogenesis. Our previous study had demonstrated that TvFACP down-regulates F-actin assembly, and its overexpression substantially decreased actin-based morphological transformation and cytoadherence in T. vaginalis. In this study, we generated the mutants with deletion of α-actin-binding domain (TvFACP △237) or mutation at putative CKII phosphorylation site (TvFACP S2AS4A) for functional assay. When examined by immunoprecipitation, α-actin decreased the activity of complex formation with TvFACP △237, but that little changed with TvFACP S2AS4A. Meanwhile, F-actin polymerization inhibited by overexpression of TvFACP was partially restored in the mutants overexpressing △237 and S2AS4A. Additionally, solid-phase plate binding and co-sedimentation assays demonstrated that TvFACP could bind F-actin and G-actin with similar strength to co-regulate α-actin polymerization in T. vaginalis. Intriguingly, TvFACP overexpression accelerated migratory behavior of trophozites in a modified trans-well assay. All data together, we speculate that TvFACP directly binds with α-actin mommer or polymer to regulate F-actin assembly leading to the changes of morphogenesis, cytoadherence and migration in this parasite.

並列關鍵字

Trichomonas vaginalis ； F-actin assembly ； F-actin capping protein ； α-actin-binding domain ； CKII phosphorylation sites

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