Sortase mediated protein ubiquitination with defined chain length and topology.
Sortase mediated protein ubiquitination with defined chain length and topology.
复制标题
分选酶介导的蛋白质泛素化具有确定的链长度和拓扑结构。
DOI：
10.1039/d3cb00229b
复制
发表时间：
2024
影响因子：
4.1
通讯作者：
Burslem,GeorgeM
中科院分区：
文献类型：
--
作者：
Raniszewski,NicoleR;Beyer,JennaN;Noel,MylesI;Burslem,GeorgeM
泛素化是蛋白质赖氨酸侧链翻译后的一个关键修饰，已知会影响蛋白质稳定性、信号转导级联、蛋白质-蛋白质相互作用等。在开发新的方法来产生多泛素的离散链并将它们特异地结合到蛋白质上方面已经取得了很大的进展，方法包括化学合成方法、酶的方法以及两者之间的许多方法。先前的工作已经证明了细菌转肽酶分选酶（SrtA）的工程变体在泛素位点特异性结合到靶蛋白上的效用。在这篇文章中，我们将经典的E1/ e2介导的多泛素链延伸方法与排序酶介导的连接和点击化学相结合，使单、二和三泛素化蛋白sfGFP和PCNA的产生成为可能。我们证明了这种策略的实用性，以产生k48连接和k63连接的多泛素，并将它们连接到n端和位点特异性的蛋白质上。此外，我们强调了两种常用的分选酶变体SrtA 5M和7M之间的活性差异，并证明虽然SrtA 7M可用于将这些泛素结合到底物上，但SrtA 5M可用于从底物中释放泛素以及从所使用的泛素变体中切割c末端标签。总的来说，我们设想这种方法广泛适用于容易产生离散的多泛素链的任何连锁类型，可以通过E1/E2系统访问，并特异性地偶联到感兴趣的蛋白质上，从而获得定制的泛素化蛋白质，这是目前不可能的。
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.