- 學位論文
SUMO1自我長鏈聚合之研究與SENPs降解多SUMO長鏈 之性質分析
Study of the Self-polymerization of SUMO1 and Characterization of the PolySUMO Chain Degradation Catalyzed by SENPs
摘要
透過SUMO化修飾目標蛋白質或以 SUMO 專一性蛋白酶 (SUMO/Sentrin-specific proteases, SENPs) 進行其可逆性移除乃是細胞內重要的蛋白質修飾作用。與泛素化修飾相同，SUMO 化修飾亦調控了細胞中許多重要功能，包括影響蛋白質間之交互作用與穩定性等。已知泛素可透過不同位置形成聚合長鏈，且其具有不同的生理意義，而 SUMO 化修飾雖被證實亦可形成多 SUMO 長鏈，然其生理角色與形成機制仍不十分清楚。 本論文透過將 SUMO 化修飾所需之各酵素轉形至大腸桿菌中，建立一不受 SENPs 所調節的 SUMO 化修飾反應系統，進行多 SUMO 化修飾的觀察與探討。結果顯示，在所建立的系統中，被認為無法形成聚合長鏈的 SUMO1 可透過非保守性 SUMO 化修飾作用於特定離胺酸形成聚合長鏈，且其生成的位置偏好發生於結構鬆散的 N 端，顯示空間上的障礙乃是影響多 SUMO 長鏈生成的重要因素。為探討各 SENP 對於多 SUMO 長鏈降解之影響，本論文以細胞外 SENP 活性分析發現，除 SENP5 外，各 SENP 其 C 端蛋白酶活性區皆可水解多 SUMO1 及多 SUMO2 長鏈，而其中 SENP1、SENP2 水解兩種 SUMO 長鏈之活性並無顯著差異。由於先前研究多認為 SENP1 及 SENP2 不具有降解多 SUMO 長鏈的能力，本論文所得結果顯示降解多 SUMO 長鏈的活性廣泛存在於大多數 SENP 保守性的活性區中。此外，本研究亦發現 SENP6、SENP7 蛋白酶活性區水解多 SUMO2 長鏈的活性較多 SUMO1 為佳，顯示其活性區與受質專一性有關。
並列摘要
The conjugation of SUMO moiety by SUMO conjugation system and the removal of the moiety by SENPs are important aspects of protein modification in cells. Analogous to ubiquitination, sumoylation regulates many physiological functions of proteins. It has been demonstrated that ubiquitin can form various polymeric chains via specific lysines to execute distinct functions. Although certain types of polymeric SUMO chains have been described, their roles in biological processes and the mechanism of formation remain unclear. In this study, I transformed the enzymes required for sumoylation into Escherichia coli BL21 (DE3) to establish a sumoylation system, which lacks SENP-mediated desumoylation for analyzing the formation of polymeric SUMO chains. The results suggest that SUMO1, which has been considered unable to form polymers, is able to form polymeric chains mainly through self-conjugation at specific lysine residues nearby the flexible N terminus, indicating that the steric hindrance is a key factor modulating polySUMO chains formation. To investigate the character of polySUMO degradation catalyzed by SENPs, I examined the degradation of polySUMO chains by conducting the in vitro SENP activity analysis. The data showed that the catalytic domains of all SENPs, except SENP5, are able to deconjugate both polySUMO1 chains and polySUMO2 chains. Moreover, the catalytic core of SENP1 and SENP2 present similar preference toward polySUMO1 and polySUMO2 chains. I also found that the catalytic cores of SENP6 and SENP7 exhibit an exquisite substrate selectivity that polySUMO2 chains are deconjugated more efficiently than polySUMO1 chains.
參考文獻
延伸閱讀
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