一种可扩展的、基于细胞的 Ube3a 变体功能评估方法
Summary
开发了一种简单且可扩展的方法来评估Ube3a中错义变异的功能意义， Ube3a是一种基因，其功能的丧失和获得与Angelman综合征和自闭症谱系障碍有关。
Abstract
测序在医学中的使用越来越多，已经在人类基因组中发现了数百万个编码变异。这些变异中的许多发生在与神经发育障碍相关的基因中，但绝大多数变异的功能意义仍然未知。本协议描述了Ube3a变异的研究， Ube3a是一种编码与自闭症和安格曼综合征相关的E3泛素连接酶的基因。 Ube3a 的重复或三重与自闭症密切相关，而其缺失会导致Angelman综合征。因此，了解UBE3A蛋白活性变化的效价对临床结果很重要。在这里，描述了一种快速的，基于细胞的方法，该方法将 Ube3a变体与Wnt 通路报告基因配对。这种简单的测定是可扩展的，可用于确定任何 Ube3a 变体中活性变化的价和幅度。此外，该方法的便利性允许产生丰富的结构功能信息，这些信息可用于深入了解UBE3A的酶机制。
Introduction
最近的技术进步使外显子组和基因组的测序成为临床环境中的常规1，2。这导致了大量遗传变异的发现，包括数百万个错义变异，这些变异通常会改变蛋白质中的一个氨基酸。了解这些变异的功能意义仍然是一个挑战，只有一小部分（~2%）已知的错义变异具有临床解释1，3。
这个问题的一个突出例子是Ube3a，这是一种编码E3泛素连接酶的基因，该酶靶向底物蛋白进行降解4。Ube3a 位于染色体 15q11-13 内，仅由母系遗传等位基因5，6，7 表达。疾病遗传学的观察强烈表明，UBE3A酶活性不足或过度会导致明显的神经发育障碍。母体染色体 15q11-13 缺失会导致安格曼综合征 （AS）8，这是一种以严重智力障碍、运动障碍、癫痫发作、快乐举止和频繁微笑和面部畸形特征为特征的疾病8，9，10。相比之下，母体染色体15q11-13的重复或三重会导致Dup15q综合征，这是一种被认为是自闭症最普遍的综合征形式之一的异质性疾病11，12，13。此外，在Ube3a中发现了数百种错义变异，其中大多数被认为是具有不确定意义的变异（VUS），因为它们的功能和临床意义是未知的。因此，人们对开发能够实证评估Ube3a变异以确定它们是否有助于神经发育疾病的方法产生了相当大的兴趣。
UBE3A 酶属于 E3 泛素连接酶的 HECT（与 E6-AP C 末端同源）结构域家族，它们都具有同名的 HECT 结构域，其中包含接受来自 E2 酶的活化泛素并将其转移到底物蛋白所需的生化机制14。从历史上看，E3酶的表征依赖于需要纯化蛋白质集合的体外重建系统4，15，16。这种方法缓慢且费力，不适合评估大量变体的活性。在以前的工作中，UBE3A被鉴定为通过调节蛋白酶体的功能来激活HEK293T细胞中的Wnt途径，以减缓β-连环蛋白17的降解。这种洞察力允许使用 Wnt 通路报告基因作为一种有效和快速的方法来识别 Ube3a18 的功能丧失和获得变体。下面的协议详细描述了一种生成Ube3a变体的方法以及基于荧光素酶的报告基因，用于评估Ube3a变体活性的变化。
Protocol
1. 诱变克隆生成Ube3a 变体 将所有 Ube3a 变体克隆到pCIG2质粒中（图1A），这是一种双纤载体，其中包含鸡-β-肌动蛋白启动子和用于EGFP表达的内部核糖体进入位点（IRES）19。全长 Ube3a 构建体包含一个N端Myc标签序列，并使用5’SacI位点和3’XmaI位点克隆到pCIG2中。此外， Ube3a 编码序列中天然存在的EcoRI，EcoRV和PstI位点?…
Representative Results
Ube3a错义变异的大规模功能筛选确定了功能丧失和获得突变的广泛前景先前对Ube3a突变体的研究表明，Wnt反应可以作为细胞 UBE3A 蛋白活性的报告基因。扩大了这些观察结果，并进行了额外的验证实验，以研究BAR测定是否适合报告细胞中的一系列UBE3A活性。首先，用编码人WT Ube3a的不同量的质粒DNA转染HEK293T细胞。该实验表明，BAR反应随转染到细胞中的 Ube3a ?…
Discussion
此处描述的方案提供了一种有效且可扩展的方法来评估Ube3a变体的酶活性。使用该测定时，有几个技术细节值得仔细考虑。一个考虑因素是选择该测定中使用的Wnt报告质粒。这里描述的方案专门使用β-连环蛋白激活报告基因（BAR）21，该报告基因包含由专门设计的接头序列分隔的12个T细胞因子（TCF）响应元件的串联体，以最大限度地减少TCF结合位点的重组和丢失。文献中还…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了西蒙斯基金会独立之桥奖（SFARI奖#387972;J.J.Y.），大脑和行为研究基金会（J.J.Y.）的NARSAD青年研究员奖，阿尔弗雷德·P·斯隆基金会（J.J.Y.）的研究奖学金，以及天使综合症基金会（J.J.Y.），白厅基金会（J.J.Y.）和NIMH（R01MH122786;杰杰）。
Materials
| 0.05% Trypsin-EDTA (1x), phenol red | Gibco | 25300-054 | |
| 1 Kb DNA ladder | Lambda Biotech | M108-S | |
| 100 bp DNA Ladder | Lambda Biotech | M107 | |
| 10x Buffer for T4 DNA Ligase with 10 mM ATP | New England BioLabs | B0202A | |
| 5x Phusion HF Reaction Buffer | New England BioLabs | B0518S | |
| Antibiotic-Antimycotic Solution | Corning | 30004CI | |
| Black/White Isoplate-96 Black Frame White Well plate | PerkinElmer | 6005030 | |
| Carbenicillin Disodium Salt | Midwest Scientific | KCC46000-5 | |
| Countess cell counting chamber slides | Invitrogen by Thermo Fisher Scientific | C10283 | |
| Countess II Automated Cell Counter | life technologies | Cell counting machine | |
| Custom DNA oligos | Integrated DNA Technologies (IDT) | ||
| Deoxynucleotide (dNTP) Solution Mix | New England BioLabs | N0447S | |
| DMEM, high glucose, GlutaMAX Supplement, pyruvate | Gibco | 10569044 | Basal medium for supporting the growth of HEK293T cell line |
| DPBS (1x) | Gibco | 14190-136 | |
| Dual-Luciferase Reporter Assay System | Promega | E1910 | |
| EcoRI-HF | New England BioLabs | R3101S | Restriction enzyme |
| Fetal Bovine Serum, qualified, heat inactivated | Gibco | 16140071 | Fetal bovine serum |
| Fisherbrand Surface Treated Tissue Culture Dishes | Fisherbrand | FB012924 | |
| FuGENE 6 Transfection Reagent | Promega | E2691 | |
| Gel Loading Dye Purple (6x) | New England BioLabs | B7024A | |
| HEK293T cells | ATCC | CRL-3216 | |
| High Efficiency ig 10B Chemically Competent Cells | Intact Genomics | 1011-12 | E. coli DH10B cells |
| HiSpeed Plasmid Midi Kit | Qiagen | 12643 | Midi prep |
| pCIG2 plasmid | |||
| pGL3 BAR plasmid | |||
| Phusion HF DNA Polymerase | New England BioLabs | M0530L | DNA polymerase |
| ProFlex 3 x 32 well PCR System | Applied biosystems by life technologies | Thermocycler | |
| pTK Renilla plasmid | |||
| QIAprep Spin Miniprep Kit (250) | Qiagen | 27106 | Mini prep |
| QIAquick Gel Extraction Kit (250) | Qiagen | 28706 | Gel purification |
| QIAquick PCR Purification Kit (250) | Qiagen | 28106 | PCR purification |
| rCutSmart Buffer | New England BioLabs | B6004S | |
| SacI-HF | New England BioLabs | R3156S | Restriction enzyme |
| Synergy HTX Multi-Mode Reader | BioTek | Plate reader runs Gen5 software v3.08 (BioTek) | |
| T4 DNA Ligase | New England BioLabs | M0202L | Ligase |
| TAE Buffer, Tris-Acetate-EDTA, 50x Solution, Electrophoresis | Fisher Scientific | BP13324 | |
| Tissue Culture Plate 96 wells, Flat Bottom | Fisherbrand | FB012931 | |
| UltraPure Ethidium Bromide Solution | Invitrogen by Thermo Fisher Scientific | 15585011 | |
| XmaI | New England BioLabs | R0180S | Restriction enzyme |
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Citar este artículo
Stelzer, J. A., Yi, J. J. A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants. J. Vis. Exp. (188), e64454, doi:10.3791/64454 (2022).