A new FRET-based platform to track substrate ubiquitination by fluorescence.
A new FRET-based platform to track substrate ubiquitination by fluorescence.
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DOI：
10.1074/jbc.ra120.016858
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Pan ZQ
中科院分区：
文献类型：
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作者：
Wu K;Ching K;Chong RA;Pan ZQ
泛素（Ub）对蛋白质的翻译后修饰改变了靶蛋白的稳定性、亚细胞位置或功能，从而影响了许多生物过程，并直接导致了无数的细胞缺陷或疾病状态，如癌症。通过荧光跟踪底物泛素化为高级反应动力学研究和包括药物发现在内的转化研究提供了机会。然而，泛素化研究中基于荧光的技术仍未得到充分开发，至少部分原因是与Ub链复杂性和对额外底物修饰的要求相关的挑战。在这里，我们描述了一个一般的策略，FRET二泛素化，跟踪底物泛素化荧光。该平台根据底物与其同源E3 Ub连接酶之间的特定相互作用产生均匀的双Ub产物。二泛素化分别在ub连接的供体和受体荧光团之间产生接近，使能量转移产生独特的荧光信号。FRET二泛素化依赖于ub -底物融合，这可以使用两种有效的策略之一来实现。方法1是使用重组底物- ub融合，适用于所有能与E3结合的底物肽。方法2是一种化学酶连接方法，利用合成化学将Ub与含有所需修饰的底物肽融合。总之，我们新的基于fret的二泛素化系统提供了一种及时的技术，有潜力推进基础研究和翻译科学。
Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence-based techniques in ubiquitination studies remain underexplored at least partly because of challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, FRET diubiquitination, to track substrate ubiquitination by fluorescence. This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The diubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET diubiquitination relies on Ub–substrate fusion, which can be implemented using either one of the two validated strategies. Method 1 is the use of recombinant substrate–Ub fusion, applicable to all substrate peptides that can bind to E3. Method 2 is a chemoenzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based diubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.