体外 E3泛素连接酶功能分析
Summary
本研究为分析E3泛素连接酶催化活性提供了详细的 体外 泛素化测定方案。重组蛋白使用原核系统（如 大肠 杆菌培养物）表达。
Abstract
泛素（Ub）与底物蛋白的内部赖氨酸残基的共价附着，这一过程称为泛素化，代表了真核生物中最重要的翻译后修饰之一。泛素化由三种酶类的顺序级联介导，包括泛素激活酶（E1酶），泛素偶联酶（E2酶）和泛素连接酶（E3酶），有时还有泛素链伸长因子（E4酶）。在这里，提供了用于泛素化测定 的体外 方案，允许评估E3泛素连接酶活性，E2-E3对之间的合作以及底物选择。可以通过监测游离多泛素链的产生和/或E3连接酶的自动泛素化来筛选合作的E2-E3对。底物泛素化通过E3连接酶的选择性结合来定义，并且可以通过 体外 反应的蛋白质印迹来检测。此外，还描述了E2~Ub放电测定法，这是直接评估功能E2-E3合作的有用工具。在这里，泛素的E3依赖性转移从相应的E2酶跟随到游离赖氨酸氨基酸（模仿底物泛素化）或E3连接酶本身的内部赖氨酸（自泛素化）。总之，提供了三种不同的 体外 方案，这些方案快速且易于执行，以解决E3连接酶催化功能。
Introduction
泛素化是Ub与底物蛋白1共价连接的过程。Ub修饰由连续的酶促反应催化，涉及三种不同酶类的作用，即Ub激活酶（E1s），Ub偶联酶（E2s），Ub连接酶（E3s）和可能的Ub链伸长因子（E4s）2，3，4，5。在三磷酸腺苷（ATP）和镁（Mg2+）依赖性Ub被E1激活后，E1的活性位点半胱氨酸攻击Ub的C端甘氨酸，形成硫酯复合物（Ub~E1）。从ATP水解中汲取的能量使Ub达到高能量过渡状态，该状态在随后的酶级联中保持。接下来，E2酶将活化的Ub转移到其内部催化半胱氨酸中，从而形成瞬态Ub~E2硫酯键。随后，Ub被转移到底物蛋白上。
这可以通过两种方式完成。E3连接酶可以首先与E2结合，或者E3连接酶可以直接结合Ub。后一种方式导致E3~Ub中间体的形成。在任何一种情况下，Ub通过在Ub的C端羧基和底物6的赖氨酸Ɛ-氨基之间形成同肽键与底物蛋白连接。人类基因组编码两个E1，大约40个E2和600多个推定的泛素连接酶7。基于E3的Ub转移机制，将Ub连接酶分为三类，涉及E6AP C-Terminus（HECT）型同源，真正有趣的新基因（RING）/U-box型，以及RING（RBR）型连接酶8之间的RING。在这项研究中，含有连接酶的U盒，HSC70相互作用蛋白（CHIP）的羧基终点，被用作代表性的E3酶。与形成Ub~E3硫代酯的HECT型E3酶相反，CHIP的U-box结构域结合E2~Ub并促进随后的Ub/底物直接从E2酶8，9转移。基于U型箱对酶促功能的重要性，使用非活性的芯片U型箱突变体CHIP（H260Q）作为对照。CHIP（H260Q）无法与其同源E2s结合，从而失去其E3连接酶活性10。
蛋白质泛素化在调节真核细胞中的多种细胞事件中起着至关重要的作用。Ub分子与底物蛋白的可逆附着促进的细胞结果的多样性可归因于Ub的分子特性。由于Ub本身含有七个赖氨酸（K）残基，用于进一步泛素化，因此存在丰富多样的具有不同尺寸和/或拓扑结构的Ub链类型11。例如，底物可以被单个Ub分子在一个（单泛素化）或多个赖氨酸（多单泛素化）修饰，甚至通过Ub链（多泛素化）11。Ub链通过相同或不同的Ub赖氨酸残基形成同型或异型，这甚至可能导致支链Ub链9。因此，蛋白质泛素化导致Ub分子的多种排列，提供特异性信息，例如，用于偶联蛋白的降解，活化或定位12，13。这些不同的Ub信号能够快速重编程细胞信号通路，这是细胞响应不断变化的环境需求能力的重要要求。
泛素化的一个核心方面与蛋白质质量控制有关。错误折叠或不可逆受损的蛋白质必须被降解并被新合成的蛋白质取代，以维持蛋白质稳态或蛋白质稳态14。质量控制E3连接酶，CHIP，与分子伴侣合作，在受损蛋白9，15，16，17的Ub依赖性降解中合作。除此之外，CHIP调节肌球素导向的伴侣UNC-45B（Unc-45同源物B）的稳定性，其与肌肉功能紧密协调，偏离最佳水平导致人肌病18，19，20，21。26S蛋白酶体对UNC-45B的降解是由K48连接的聚Ub链9的附着介导的。在没有底物蛋白的情况下，CHIP进行自泛素化10，22，23，这是RING/ U-box E3泛素连接酶24，25的特征，并被认为调节连接酶活性26。本文中描述的体外泛素化测定方法的应用有助于系统地鉴定与CHIP合作促进游离聚Ub链形成和/或CHIP的自动泛素化的E2酶（方案部分2）。此外，观察到UNC-45B的CHIP依赖性泛素化，这是E3连接酶18，19的已知底物（方案部分3）。最终，监测了来自Ub~E2硫酯的活化Ub的CHIP依赖性转移（协议第4节）。
Protocol
Representative Results
Discussion
本文介绍了用于分析E3连接酶功能的基本 体外 泛素化方法。在进行 体外 泛素化测定时，应考虑一些E2酶可以进行自泛素化，因为它们的活性半胱氨酸攻击其自身的赖氨酸残基，这些赖氨酸残基位于活性位点30附近。为了规避这个问题，建议使用E2突变体，其中将相应的赖氨酸残基交换为精氨酸，这导致催化活性E2酶对自身泛素化32具有抗性。当通过…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢实验室成员对手稿的批判性讨论和有益的建议。由于大小限制，我们很抱歉没有引用有价值的贡献。这项工作得到了Deutsche Forschungsgemeinschaft（DFG，德国研究基金会）的支持 – SFB 1218 – Projektnumber 269925409和卓越集群EXC 229 / CECAD到TH。这项工作由德国卓越战略（EXC 2030 – 390661388和SFB 1218 – Projektnumber 269925409到T.H. Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft（DFG）资助，用于德国卓越战略 – EXC 2030 – 390661388 und – SFB 1218 – Projektnummer：269925409 an T.H. gefördert。
Materials
| Amershan Protran 0.1 µm NC | GE Healthcare | 10600000 | nitrocellulose membrane |
| Anti-CHIP | Cell Signaling | 2080 | Monoclonal rabbit anti-CHIP antibody, clone C3B6 |
| Anti-MYC | Roche | OP10 | Monoclonal mouse anti-MYC antibody, clone 9E10 |
| Anti-ubiquitin | Upstate | 05-944 | Monoclonal mouse anti-Ub antibody, clone P4D1-A11 |
| Apyrase | Sigma | A6535-100UN | |
| ATP (10x) | Enzo | 12091903 | |
| BSA | Sigma | A6003-10G | |
| EDTA | Roth | 8043.2 | |
| KCl | Roth | 6781.1 | |
| K2HPO4 | Roth | P749.2 | |
| KH2PO4 | Roth | 3904.1 | |
| LDS sample buffer (4x) | novex | B0007 | |
| L-Lysine | Sigma | L5501-5G | |
| MES | Roth | 4256.4 | |
| MeOH | VWR Chemicals | 2,08,47,307 | 100% |
| Milchpulver | Roth | T145.3 | |
| NaCl | Roth | P029.3 | |
| NuPAGE Antioxidant | invitrogen | NP0005 | |
| NuPAGE Transfer buffer (20x) | novex | NP0006-1 | |
| Page ruler plus | Thermo Fisher | 26619 | Protein ladder |
| RotiBlock | Roth | A151.1 | Blocking reagent |
| SDS (20%) | Roth | 1057.1 | |
| S1000 Thermal Cycler | Bio Rad | 1852196 | |
| Trans-Blot Turbo | Bio Rad | 1704150EDU | Transfer system |
| Tris base | Roth | 4855.3 | |
| Tween 20 | Roth | 9127.2 | |
| UbcH Enzyme Set | BostonBiochem | K-980B | E2 enzymes |
| Ubiquitin | BostonBiochem | U-100H | |
| Ubiquitin-activating enzyme E1 | Enzo | BML-UW941U-0050 | |
| Ubiquitylation buffer (10x) | Enzo | BML-KW9885-001 | |
| Whatman blotting paper | Bio Rad | 1703969 | Extra thick filter paper |
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