Examination of the Deubiquitylation Site Selectivity of USP51 by Using Chemically Synthesized Ubiquitylated Histones
Examination of the Deubiquitylation Site Selectivity of USP51 by Using Chemically Synthesized Ubiquitylated Histones
复制标题
使用化学合成的泛素化组蛋白检查 USP51 的去泛素化位点选择性
DOI：
10.1002/cbic.201800432
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发表时间：
2019-01-18
期刊：
影响因子：
3.2
通讯作者：
Liu, Lei
中科院分区：
文献类型：
--
作者：
Ai, Huasong;Guo, Yu;Liu, Lei
组蛋白泛素化和去泛素化过程及其调控机制与表观遗传学密切相关。最近，据报道去泛素化酶USP 51选择性地切割组蛋白H2 A上K13或K15处的泛素化（即，H2 AK 13 Ub和H2 AK 15 Ub），但在K119处没有（即，H2AK119Ub）。为了阐明USP 51的选择性机制，我们构建了结构明确的体外蛋白质系统，在精确的位点进行泛素修饰。开发了一种全化学蛋白质合成方法，其中使用基于酰肼的天然化学连接来有效地产生五种泛素化组蛋白（H2 AK 13 Ub、H2 AK 15 Ub、H2 AK 119 Ub、H2 BK 34 Ub和H2 BK 120 Ub）。将这些合成的泛素化组蛋白组装成核小体，并进行体外USP 51去泛素化测定。令人惊讶的是，与先前的体内观察相反，USP 51没有显示出H2 AK 13/15 Ub和H2 AK 119 Ub之间的偏好。因此，对USP 51选择性的理解可能需要考虑其他因素，例如替代的预先存在的组蛋白修饰、竞争性阅读蛋白或体内提取核小体和体外重构核小体之间的不同核小体质量。进一步的实验证实，USP 51在体外可以使携带H2 BK 120 Ub的核小体去泛素化，但不能使携带H2 BK 34 Ub的核小体去泛素化。分子动力学模拟表明，USP 51催化的泛素化核小体的水解受到异肽键的空间位阻的影响。
Histone ubiquitylation and deubiquitylation processes and the mechanisms of their regulation are closely relevant to the field of epigenetics. Recently, the deubiquitylating enzyme USP51 was reported to selectively cleave ubiquitylation on histone H2A at K13 or K15 (i.e., H2AK13Ub and H2AK15Ub), but not at K119 (i.e., H2AK119Ub), in nucleosomes in vivo. To elucidate the mechanism for the selectivity of USP51, we constructed structurally well-defined in vitro protein systems with a ubiquitin modification at precise sites. A total chemical protein synthesis procedure was developed, wherein hydrazide-based native chemical ligation was used to efficiently generate five ubiquitylated histones (H2AK13Ub, H2AK15Ub, H2AK119Ub, H2BK34Ub, and H2BK120Ub). These synthetic ubiquitylated histones were assembled into nucleosomes and subjected to in vitro USP51 deubiquitylation assays. Surprisingly, USP51 did not show preference between H2AK13/15Ub and H2AK119Ub, in contrast to previous in vivo observations. Accordingly, an understanding of the selectivity of USP51 may require consideration of other factors, such as alternative pre-existing histone modifications, competitive reader proteins, or different nucleosome quality among the in vivo extraction nucleosome and the in vitro reconstitution one. Further experiments established that USP51 in vitro could deubiquitylate a nucleosome carrying H2BK120Ub, but not H2BK34Ub. Molecular dynamics simulations suggested that USP51-catalyzed hydrolysis of ubiquitylated nucleosomes was affected by steric hindrance of the isopeptide bond.