Sortase mediated protein ubiquitination with defined chain length and topology†

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Nicole R. Raniszewski, Jenna N. Beyer, Myles I. Noel and George M. Burslem
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引用次数: 0

Abstract

Ubiquitination is a key post-translational modification on protein lysine sidechains known to impact protein stability, signal transduction cascades, protein–protein interactions, and beyond. Great strides have been made towards developing new methods to generate discrete chains of polyubiquitin and conjugate them onto proteins site-specifically, with methods ranging from chemical synthetic approaches, to enzymatic approaches and many in between. Previous work has demonstrated the utility of engineered variants of the bacterial transpeptidase enzyme sortase (SrtA) for conjugation of ubiquitin site-specifically onto target proteins. In this manuscript, we’ve combined the classical E1/E2-mediated polyubiquitin chain extension approach with sortase-mediated ligation and click chemistry to enable the generation of mono, di, and triubiquitinated proteins sfGFP and PCNA. We demonstrate the utility of this strategy to generate both K48-linked and K63-linked polyubiquitins and attach them both N-terminally and site-specifically to the proteins of interest. Further, we highlight differential activity between two commonly employed sortase variants, SrtA 5M and 7M, and demonstrate that while SrtA 7M can be used to conjugate these ubiquitins to substrates, SrtA 5M can be employed to release the ubiquitin from the substrates as well as to cleave C-terminal tags from the ubiquitin variants used. Overall, we envision that this approach is broadly applicable to readily generate discrete polyubiquitin chains of any linkage type that is accessible via E1/E2 systems and conjugate site-specifically onto proteins of interest, thus granting access to bespoke ubiquitinated proteins that are not currently possible.

Abstract Image

Abstract Image

分类酶介导的蛋白质泛素化具有明确的链长和拓扑结构
泛素化是蛋白质赖氨酸侧链上的一种关键的翻译后修饰,已知会影响蛋白质的稳定性、信号转导级联、蛋白质之间的相互作用等。在开发新方法以生成离散的多泛素链并将其特异性地连接到蛋白质上方面取得了长足进步,这些方法包括化学合成方法、酶解方法以及许多介于两者之间的方法。以前的工作已经证明,细菌转肽酶的工程变体 SrtA 可以将泛素定点连接到目标蛋白质上。在本手稿中,我们将经典的 E1/E2 介导的多泛素链延伸方法与分选酶介导的连接和点击化学结合起来,生成了单泛素化、双泛素化和三泛素化蛋白质 sfGFP 和 PCNA。我们展示了这种策略在生成 K48 连接和 K63 连接的多泛素并将其 N 端和特定位点连接到相关蛋白质上的实用性。此外,我们还强调了两种常用分选酶变体(SrtA 5M 和 7M)之间的不同活性,并证明 SrtA 7M 可用于将这些泛素与底物连接,而 SrtA 5M 则可用于从底物中释放泛素,以及从所用泛素变体中裂解 C 端标签。总之,我们设想这种方法可广泛应用于通过 E1/E2 系统随时生成任何连接类型的离散多泛素链,并按特定位点连接到感兴趣的蛋白质上,从而获得目前不可能获得的定制泛素化蛋白质。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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