芋頭半胱胺酸蛋白酶抑制因子之生物資訊及生化分析

Domain analysis of tarocystatin by bioimformatic and biochemical assay

朱明宏(Ming-Hung Chu)

指導教授 ： 鄭貽生

國立臺灣大學/生命科學院/植物科學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00574

摘要

芋頭半胱胺酸蛋白酶抑制因子(tarocystatin)屬於第二群植物半胱胺酸蛋白酶抑制因子(phytocystatins)。Phytocystatins相似之處在於均具有高度保守的胺基端區域(N-terminal domain, NtD)，稱之為cystatin domain (CY domain)。不同的是第二群抑制因子比第一群在蛋白質羧基端多了胺基酸序列的延伸，但此羧基端區域(C-terminal domain, CtD)功能尚不明確。為確認NtD與CtD能否產生交互作用，藉由戊二醛交聯反應(glutaraldehyde corss-linking)及酵母菌雙雜交實驗(yeast two-hybrid)的生化方式進行分析，結果顯示NtD與CtD具有微弱的交互作用，且NtD彼此間亦會產生交互作用，使tarocystatin能形成homodimer。
由胺基酸序列比對發現NtD、CtD與已知蛋白質結構的水稻半胱胺酸蛋白酶抑制因子(oryzacystatin, OC-Ⅰ)在序列上有相似性，而蛋白質二級結構預測也發現NtD與CtD的組成相似，均由1個α-helix緊接著4個β-sheets所組成，推測CtD蛋白質的摺疊方式應與NtD類似，稱之為類CY區域(CY-like domain)，而此種二級結構組成方式亦和OC-I相同。因此以OC-Ⅰ為模板，根據交互作用實驗的結果，以同源模擬法(homology modeling)和蛋白質對接(protein-protein docking)的方式建構出兩種不同型式的tarocystatin之全長結構。
在功能分析方面，由膠體抑制活性染色實驗確認CtD具有微弱促進木瓜酶活性的能力，而全長抑制木瓜酶的效果比NtD強；又GST融合之tarocystatin會加強原始蛋白的功效，造成GST-CtD促進木瓜酶活性的能力優於CtD，而GST-全長和GST-NtD的抑制效果更佳。由本實驗的結果推論，在正常狀況下tarocystatin會以單體(monomer)及同型雙體(homodimer)的形式共存，而CtD的出現可能扮演促進tarocystatin抑制木瓜酶活性之功能。

關鍵字

半胱胺酸蛋白酶抑制因子 ； 芋頭 ； 同源模擬法 ； 蛋白質對接

並列摘要

Tarocystatin is a member of group-2 phytocystatin which could help plants against insect and pathogen attack. It shares a similar cystatin domain in the N-terminal region (NtD), and contained an extended C-terminal domain (CtD). For identifying the interaction of NtD and CtD, the glutaraldehyde cross-linking and yeast two-hybrid assays were carried out, the results showed a weak interaction between NtD and CtD, and tarocystatin could form homodimer by interaction of NtD and NtD. According to amino acids sequence alignment and secondary structure prediction, the fold of CtD should be a cystatin-like domain (CY-L domain) with similar folding to NtD which is 1 α-helix followed by 4 β-sheets. Based on domain interaction and bioinformatic analyses, we performed homology modeling and protein-protein docking to build the two models of tarocystatin using OC-I as the template. Moreover, we confirmed that CtD possess weak papain activitation property by in-gel inhibitory activity assay, full-length of tarocystatin showed higher anti-papain activity than NtD, and the GST fused tarocystatin augment their original property than none fused protein. As the results, we speculated that native tarocystatin could exist both monomeric and homodimeric form, and CtD might play a role in enhancing the inhibitory activity of tarocystatin.

並列關鍵字

cysteine protease inhibitor ； taro ； homology modeling ； protein-protein docking

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