建立以單分子螢光顯微技術研究Shugoshin-1蛋白與潛力治療用胜肽間動態交互作用的平台

Development of a single-molecule-fluorescence-microscopy-based platform to study dynamic interactions between Shugoshin-1 and potential therapeutic peptides

陳彥霖(Chen Yan-Lin)

指導教授 ： 李以仁

國立臺灣師範大學/理學院/化學系/碩士(2021年)

https://doi.org/10.6345/NTNU202101171

摘要

蛋白質-蛋白質交互作用(Protein-Protein interactions, PPIs)促進大部分生物機能，蛋白質區域與區域之間的交互作用(Domain-Domain interactions, DDIs)引導功用性蛋白在特定區域作用，交互作用必須通過蛋白質的二級結構進行，二級結構中螺旋結構穩定且單純，探討螺旋結構之間的交互作用能夠得到更精確的結果。
Shugoshin 1 (Sgo1)和蛋白磷酸酶2A (PP2A)之間的結合研究說明Sgo1募集PP2A在保護姊妹染色體中有重要的作用。為了發展一系列抗微生物胜肽，我們擷取Sgo1-PP2A交互作用的片段，將目標Sgo1胜肽和DNA交聯，並經由與生物素標記的互補DNA結合而固定在單分子檢驗平台上，並在緩衝溶液中添加螢光標記的游離潛力治療用胜肽進行交互作用，利用單分子螢光顯微鏡研究胜肽-胜肽交互作用動力學。和傳統的方法比較，需要的胜肽濃度相對較低(< 100 nM)，使我們可以對潛力治療用胜肽進行更廣泛的篩選。

關鍵字

胜肽-胜肽交互作用 ； Sgo1 ； PP2A ； 抗微生物胜肽 ； 單分子螢光共振能量轉移

並列摘要

Protein-Protein Interactions (PPIs) facilitate many biological functions, and Domain-Domain Interactions (DDIs) induce functional protein activity in specific regions. The interactions between domains are usually dictated by their secondary structures . Helical structure is relatively simple and stable , hence , becomes a suitable candidate for studying domain-domain interactions . Previous study of Shugoshin 1(Sgo1)-Protein Phosphatase 2A(PP2A) binding demonstrated that recruitment of PP2A by Sgo1 plays an important role in the protection of sister chromatid , Hence , becomes a potent target for the development of antimicrobial peptides. We extract the fragments of Sgo1-PP2A interaction and establish a single-molecule-fluorescence-microscopy-based platform for studying the peptide-peptide interaction dynamics. Target peptide (Sgo1)-DNA hybrid was used in the immobilized single-molecule assays and interacted with the fluorescence-labeled free potential therapeutic peptides in buffer solution. The required concentration (< 100 nM) of peptides is relatively low compared to conventional methods, allowing us to perform a wider range of screening on the potential therapeutic peptides.

並列關鍵字

Peptide-Peptide interactions ； Sgo1 ； PP2A ； Antimicrobial peptides ； smFRET

參考文獻

[1] Huan, Y.; Kong, Q.; Mou, H.; Yi, H. Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Frontiers in Microbiology. 2020, 11, 582779

[2] Lau, J. L.; Dunn, M. K. Therapeutic Peptides: Historical Perspectives, Current Development Trends, and Future Directions. Bioorganic & Medicinal Chemistry 2018, 26 (10), 2700–2707.

[3] Marqus, S.; Pirogova, E.; Piva, T. J. Evaluation of the Use of Therapeutic Peptides for Cancer Treatment. Journal of Biomedical Science 2017, 24 (1), 21.

[4] Burba, A. E. C.; Lehnert, U.; Yu, E. Z.; Gerstein, M. Helix Interaction Tool (HIT): A Web-Based Tool for Analysis of Helix-Helix Interactions in Proteins. Bioinformatics 2006, 22 (22), 2735–2738.

[5] Kangueane, P.; Nilofer, C. Principles of Protein-Protein Interaction. In Protein-Protein and Domain-Domain Interactions; Springer Singapore: Singapore, 2018; 93–111.