Subunit-Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases.
Subunit-Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases.
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DOI：
10.1002/cbic.201600276
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发表时间：
2016-08-17
期刊：
影响因子：
--
通讯作者：
Strieter ER
中科院分区：
文献类型：
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作者：
Crowe SO;Pham GH;Ziegler JC;Deol KK;Guenette RG;Ge Y;Strieter ER
嵌入在不同泛素链中的信息由泛素结合结构域（UBDs）的蛋白质转导，并被一组被称为去泛素酶（DUBs）的水解酶清除。了解UBDs和DUBs的选择性对于解码不同泛素链的功能是必要的。这些努力的关键是获得具有特定位点荧光标记的化学定义的泛素链。构建这种分子的一种方法是利用分选酶（SrtA）进行肽连接，分选酶是一种细菌转肽酶，负责将细胞表面蛋白共价附着在细胞壁上。在这里，我们展示了SrtA在修饰泛素链的单个亚基中的效用。利用蛋白酶介导消化后n端甘氨酸暴露的泛素衍生物，我们合成了具有不同异肽键的泛素二聚体、三聚体和四聚体。然后将SrtA与荧光沉积肽底物结合使用，对链中的每个亚基进行修饰。通过构建带有荧光基团标记的单个亚基的泛素链分支，我们提供了泛素特异性蛋白酶USP15偏爱Ub三聚体的证据，但对特定的异肽链几乎没有偏好。我们的研究结果强调了在研究DUBs如何处理Ub链时，对Ub链进行亚基特异性标记的重要性。我们利用细菌转肽酶，分选酶合成了在单个亚基上用荧光基团修饰的支化泛素三聚体。使用这些新型荧光探针，我们创建了一种基于荧光的检测方法，用于监测水解泛素链分支时去泛素酶的连锁选择性。通过这个实验，我们发现去泛素酶USP15可能比它们的线性对应物更倾向于分支泛素链。
Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin-binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site-specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation using sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N-terminal glycine is unveiled after protease-mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin specific protease USP15 prefers Ub trimers, but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit-specific labeling of Ub chains when studying how DUBs process Ub chains. We synthesize branched ubiquitin trimers modified with fluorophores at individual subunits using the bacterial transpeptidase, sortase. Using these novel fluorescent probes, we have created a first-of-its-kind fluorescence based assay for monitoring the linkage selectivity of deubiquitinases when hydrolyzing branched ubiquitin chains. Using this assay we show that the deubiquitinase, USP15 may have a preference for branched ubiquitin chains over their linear counterparts.