胞外SUMO化系統之建立與多SUMO化機制之探討

Establishment of in vitro SUMOylation system and study of the mechanism of polySUMOylation

蔡佳芸(Jia-Yun Tsai)

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(2009年)

https://doi.org/10.6342/NTU.2009.10762

摘要

SUMO的全名為 small ubiquitin-like modifier，雖然在序列上只和泛素擁有約18%的相似度，其摺疊完成後之立體構形卻和泛素極度相似。SUMO亦藉由和泛素類似的作用機制與目標蛋白質進行共價結合，即 SUMO化 (SUMOylation) 過程也是經由E1活化酶、E2銜接酶和E3黏合酶三種酵素依序參與活化、銜接與黏合的步驟；但泛素化與SUMO化間存有三項顯著的差異：(1)在脊椎動物的系統中，目前已知參與泛素化作用的酵素有許多種類，但SUMO化僅有一種E1 (SAE1/SAE2 複合體) 和一種E2 (Ubc9) 參與；(2) 在許多in vitro研究中顯示，SUMO化可能不須要E3的參與，而僅藉由E1及E2的活性完成；(3) SUMO化是一個可逆的反應，可經由一類去SUMO化酵素破壞SUMO與目標蛋白質間的異胜肽鍵。此類SUMO蛋白酶亦負責SUMO蛋白質的成熟化，能將C端末尾的片段經水解去除，使SUMO露出雙甘胺酸序列 (di-glycine motif) 而能和目標蛋白質進行鍵結。相較於絕大多數經泛素修飾的蛋白質所導向的蛋白酶體降解途徑，SUMO化在細胞中已被發現有更多樣且範圍廣泛的生理功能，如訊息傳遞、蛋白質穩定性、酵素活性調控，以及細胞內分子運送等，因而使其相關的研究受到高度的重視。
相較於只有一種形式的泛素，目前發現的SUMO在不同生物間有許多種類，其中脊椎動物共擁有三種型式的SUMO (SUMO1-3)；其中SUMO-2和SUMO-3享有95%的序列相同性，卻僅和SUMO-1有約50%的序列相同，因此在目前的研究中，多把SUMO-2及SUMO-3總稱為 SUMO-2/3一類，而將SUMO-1獨立為另一類。特別的是，由於近年來發現SUMO-2/3的序列中具有一個SUMO化偏好發生的保守序列VKTE，因能夠像多泛素般產生聚合長鏈對目標蛋白質進行修飾，而SUMO-1由於缺乏此序列而僅能進行單一SUMO化；此項發現使單一SUMO化或多SUMO化各自帶有的生理意義受到極度重視，並引發廣泛的研究。
本論文實驗藉由點突變方法製造出帶有SUMO化共有基序的SUMO-1 (D15V)、SUMO-1 (D15V/K17T) 及SUMO-1 (K25E) 三種SUMO-1突變株，以及失去SUMO化共有基序的SUMO-2突變株SUMO-2 (K11A)，並建立在in vitro或在大腸桿菌中進行的SUMO化系統，以及能表現帶有tag之SUMO的動物穩定細胞株，作為研究SUMO化作用的工具，並探討SUMO化共有基序對SUMO聚合作用造成的影響。

關鍵字

蛋白質轉譯後修飾 ； 類泛素 ； 細胞生理

並列摘要

Although SUMO (small ubiquitin-like modifier) shares only 18% sequence identity with ubiquitin, its folding structure highly resembles that of ubiquitin except for the N terminal extension. In addition, SUMO conjugates to its substrates through a mechanism similar to that of ubiquitination. That is, SUMOylation is also a sequential enzyme catalytic cascade, which includes E1 activation, E2 conjugation and E3 ligation. However, there are three major differences between these two post-translational modifications: (1) in the mammalian system, there is only one SUMO E1 and one SUMO E2 compared to the many known ubiquitination enzymes; (2) many in vitro studies have shown that SUMOylation may not need the participation of E3 ligase; (3) SUMOylation can be reversed by de-SUMOylation enzymes. These enzymes are not only responsible for de-conjugation of SUMO but are also in charge of its C-terminal maturation. In contrast to ubiquitination, sumoylation does not target modified proteins for degradation, but can affect their signal transduction, stability, enzyme activity and localization showing its importance.
Higher eukaryotes express three SUMO family members, SUMO1-3. Mature SUMO-2 and SUMO-3 (referred to as SUMO2/3) are 97% identical but differ substantially from SUMO1 (~50% identity). Interestingly, SUMO2/3 contains an internal consensus motif that is missing in SUMO1 which is responsible for polychain formation. This gives rise to vast research on the physiological consequences of mono- and poly-SUMOylation.
To investigate the importance of SUMOylation consensus motif on SUMO polymerization, we have generated three different SUMO-1 mutants, SUMO-1(D15V), SUMO-1(D15V/K17T) and SUMO-1(K25E), which bear a SUMOylation site in their sequences. We also produced a SUMO-2 mutant losing the Lys11 SUMOylation residue by the same site-directed mutagenesis method. We have conducted several experiments with these SUMO mutants in our in vivo or in vitro SUMOylation systems, and successfully obtained some interesting findings among different types of poly-SUMOylation events.

並列關鍵字

SUMO

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