透過拓展遺傳密碼進行對去泛素化酵素之泛素-SUMO2異合性鍵結之專一性解碼

Expanding Genetic Code To Decipher The USP7 Specificity Towards Ubiquitin-SUMO2 Heterolinkage

彭尚(Kurkute Prashant Tabaji)

指導教授 ： 王彥士

共同指導教授 ： 王宗興(Tsung-Shing Wang)

國立臺灣大學/理學院/化學研究所/博士(2022年)

https://doi.org/10.6342/NTU202200707

摘要

在蛋白質的轉譯後修飾中，泛素化及類泛素小分子的蛋白質修飾，如SUMO，扮演著各式各樣的生物訊息傳遞。而在近期的研究中發現SUMO與泛素具有共同修飾的現象，增加了泛素化訊息的複雜性，且兩蛋白質間的交互作用尚未有完全的了解。
為了了解不同的小分子蛋白質修飾組合在生物功能上的交互作用，合成一定產量的異型聚合體在研究工作上成為了必不可少的工具。在合成各式聚泛素鏈的工作上已經發產出了經由酶催化、化學方法或半合成的多種途徑。在這些方法中，酶催化方法因為泛素合成酶E2及E3的選擇性有限，造成其應用受到限制；而在化學合成的途徑中，蛋白質產率與其技術的高難度亦造成使用上的限制，此外，化學合成方法只受用於構型可重新摺疊的蛋白質上。為了克服上述合成方法的困境，發展出了半合成的方法，重組蛋白的使用使得這項方法得以拓展開來。然而，在嚴厲的反應條件下仍然限制了這些技術只能應用於可重新摺疊的蛋白質。
因此，第一個主要目標即發展出一項能夠普遍受用的平臺，能夠使受體在自然條件下連接上泛素或其他類泛素蛋白質，且不必包含蛋白質重新折疊的過程。在此決定以包含硫醇的麥可加成反應（thiol-Michael addition reaction）探討合成含硫的異肽鍵，因為其具有高度選擇性且同時能夠在水溶性條件下進行。為了在受體上產生加成反應的接受端，利用基因擴展技術在指定位置上放入了非典型胺基酸SeCbzK，進一步接著進行氧化硒消除產生脫氫丙氨酸（Dha）。相較於其他當代的技術，這項生產Dha的策略具有更高的選擇性。而泛素或類泛素蛋白質則以E1酵素將半胱胺修飾於C端。同時也發展了藉由hydrazide進行自然化學連接法半合成的另一種C端修飾方法。這項方法不僅可以被廣泛擴展，更能使用在其他蛋白質的泛素化或SUMO修飾，且除去了保護與去保護反應，有助於提升整體的產量。
第二個目標則是了解去泛素化酶USP7的C端區域在受體選擇性與活性上所扮演的角色。並且，以包含硫基的異肽鍵連接的泛素二聚體顯示了與自然鍵結 具有相似的表現。所有八種泛素與SUMO的二聚體都成功被合成，並測試了與USP7間的作用，以達到了解其C端區域對雜交二聚體的反應活性與選擇性。這項研究將有助於了解USP7及泛素-SUMO鍵結之間的角色關係在癌症研究和藥物上的發展。
這項研究將替未來了解雜交二聚體的生物意義與在USP7的C端區域的選擇性和活性做到鋪成。此外，也提供了在自然條件下完成含位點選擇性的泛素或類泛素蛋白質修飾的方案。而相比先前的技術，這項方法更能得到較高的總產量。

關鍵字

體內嵌入非典型胺基酸 ； 泛素 ； 類泛素、 ； 去泛素化酵素 ； 硫醇麥可加成反應

並列摘要

Post-translation modification (PTM) of proteins with ubiquitin (Ub) or ubiquitin-like proteins (UBLs) for example small Ub-like modifiers (SUMO) plays a myriad role in cellular functions. The recent finding of cross-talk between SUMO and Ub pathways has increased the more complexity in Ub code. The function of cross-talk hasn’t been completely deciphered yet.
To understand the biological functions of different cross talk, it was essential to synthesize them in workable quantities. The various enzymatic, chemical, and semi-synthetic methods have been developed to synthesize various Ub chains. Enzymatic synthesis has restricted application because of the selectivity of E2 and E3 pairs. The application of chemical methods are restricted because of the low yield and only highly skilled laboratories can use this techniques. In addition, they are applicable to only refoldable proteins. To overcome this limitation, semi-synthetic methods have been developed. The use of recombinant proteins makes them scalable. However, the harsh reaction condition limited their applications to only refolding proteins.
Therefore, the first major aim was to develop a universal platform that can ligate the substrate with Ub or UBLs under native conditions precluding refolding necessity. The thiol-Michael addition reaction was decided to explore to synthesize thioether isopeptide because of its high selectivity and aqueous condition compatibility. To generate the Michael acceptor on the substrate the γ-Selenium-Carboxybenzy-L-Lysine (SeCbzK) was site selectively installed using expanding genetic code. Further, the β-selenoxide elimination yielded dehydroalanine (Dha) site selectively. This strategy of generating Dha is more selective as compared to the other contemporary methods. The Ub or UBLs like SUMO1 were modified at C-terminal with cysteamine using E1 enzyme. An alternative approach of semi-synthetic methods, hydrazide-based NCL, to C-terminal modification of Ub was also developed. This platform is not only scalable but also use to other proteins ubiquitination or SUMOylation. The lack of protection and deprotection helps to increase the overall yield.
The second aim was to understand the role of the C-terminal domain of ubiquitin specific protease (USP7) in the selectivity and activity of substrate. Further, the thioether isopeptide linked Ub dimers were shown to behave similarly to the native isopeptide bond. All eight Ub tagged SUMO2 were successfully synthesized and were interrogated with USP7 to understand the role of USP7 C-terminal domain (CTD) in activity and selectivity towards the hybrid dimers. This study will help to understand the role of USP7 and Ub-SUMO2 linkage in cancer study and drug development.
This study would pave the way to understand the biological meanings of hybrid dimers and the role of USP7 CTD in selectivity and activity. In addition, this study has also provided the protocol to synthesize the Ub or UBLs modified site-selective proteins in native conditions. The overall yield of our method is high as compared to the previous methods.

並列關鍵字

expanding genetic code ； ubiquitin ； SUMO ； ubiquitin specific protease ； thiol-Michael addition

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