單胞綠藻中類泛素蛋白質胜肽酶之身分辨認與生化特性分析

Identification and biochemical characterization of ubiquitin-like protein peptidases in Chlamydomonas reinhardtii

唐曉菁(Siao-Jing Tang)

指導教授 ： 張世宗

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02109

摘要

泛素與類泛素蛋白質修飾系統是利用類泛素胜肽酶、E1活化酶、E2銜接酶、甚至是E3黏合酶的作用，才能將這些小分子量的蛋白質修飾分子接至目標蛋白質的Lysine上。對人類、阿拉伯芥及酵母菌之類泛素蛋白質胜肽酶的相關研究，已經有相當多的文獻發表；相較之下，單胞綠藻在此方面的研究則仍未見。
因此，本研究致力於單胞綠藻 (Chlamydomonas reinhardtii) 中類泛素蛋白質胜肽酶的身分鑑定與生化性質分析。利用人類及阿拉伯芥類泛素蛋白質胜肽酶之序列對單胞綠藻資料庫進行搜尋與比對後，以專一性引子成功選殖出具有NEDD8 (neural precursor cell-expressed developmentally down-regulated 8) 專一性蛋白酶活性之蛋白質，並將其命名為C. reinhardtii deneddylase-1 (CrDEN1)。另外，本研究成功從單胞綠藻中選殖出N端為泛素與C端為NEDD8的特殊融合蛋白質C. reinhardtii Bi-ubiquitin (CrBi-Ub)。其能作為去泛素化酵素與NEDD8蛋白酶的受質。進一步利用CrBi-Ub之定位點突變及人類的USP2-core (ubiquitin-specific protease 2 catalytic core)與SENP8 (SUMO-specific protease 8) 來進行泛素與NEDD8蛋白酶酵素專一性的研究，發現第51號與72號之胺基酸對CrDEN1在專一性分辨泛素與NEDD8上扮演重要的角色。除此之外，本實驗所選殖到的Ubl-specific peptidase-279 (CrULP-279) 可能是一種SUMO蛋白酶，而未知其基質專一性的Ubl-specific peptidase-160 (CrULP-160) 則有待進一步的研究。

關鍵字：泛素、SUMO、NEDD8、類泛素蛋白質胜肽酶、SENP、CrBi-Ub

關鍵字

類泛素蛋白質胜肽酶

並列摘要

Ubiquitin and ubiquitin-like protein (Ubl) modification systems, composed of specific peptidases, E1 activating enzymes, E2 conjugating enzymes and E3 ligases, can specifically conjugate ubiquitin and Ubl to the lysine residues of target proteins. Studies of ubiquitin and Ubl-specific peptidases (USPs) in human, Arabidopsis and yeast have been reported in the literatures. However, the ubiquitin and USPs in Chlamydomonas reinhardtii have not been revealed.
This study focuses on the biochemical characterization of USPs of C. reinhardtii. By searching the C. reinhardtii gene bank with the amino acid sequences of human and Arabidopsis USPs, a gene encoding for the NEDD8 (neural precursor cell-expressed developmentally down-regulated 8)-specific peptidase has been identified and named C. reinhardtii deneddylase 1 (CrDEN1). Furthermore, a fusion protein consisting of an N-terminal ubiquitin and a C-terminal NEDD8 in a head-to-tail orientation has also been identified. Based on the sequence observation, this unique bi-ubiquitin protein was named CrBi-Ub, which can be considered as a substrate of deubiquitinating enzyme and NEDD8 protease. By using site-directed mutagenesis on CrBi-Ub and analysis of the proteolytic activities of USP2-core, SENP8 and CrDEN1, our data reveal that position 51 and 72 play crucial roles in molecular determinants of ubiquitin NEDD8 discrimination. Another USP named CrULP-279 in the study performed the activity of SUMO protease, but the CrULP-160 did not show any catalytic activity against the Ubl proteins analyzed in the experiments. The biochemical property of CrULP-160 remains unclear.

Keyword：Ubiquitin, SUMO, NEDD8, Ubl-specific peptidase, SENP, CrBi-Ub

並列關鍵字

ubiquitin-like protein peptidases

參考文獻

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