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Bioquímica

核糖体组蛋白的体外泛化和二次基质测定

Published: July 25, 2019
doi:
10.3791/59385
, , , , , , , ,
1Maisonneuve-Rosemont Hospital Research Center and Department of Medicine,University of Montréal, 2Lunenfeld-Tanenbaum Research Institute,Sinai Health System

Summary

泛化是一种翻译后修饰,在细胞过程中起着重要作用,并且通过二足化进行紧密协调。两种反应的缺陷是人体疾病的基础。我们提供使用纯化成分在体外进行泛化和二次脱体反应的协议。

Abstract

泛化是一种翻译后修饰,在各种信号通路中起着重要作用,并特别参与染色质功能和DNA相关过程的协调。这种修饰涉及几种酶的连续作用,包括E1泛基质激活、E2泛基丁结合和E3泛基质-结合酶,由二苯基苯甲醚逆转。泛化诱导蛋白质降解或蛋白质功能改变,包括酶活性调节、蛋白质-蛋白质相互作用和亚细胞定位。证明蛋白质泛化或二丁基化的关键步骤是使用纯化成分进行体外反应。有效的泛化和二次成位反应可能受到所使用的不同成分、酶共因子、缓冲条件和基质性质的影响。 在这里,我们提供用于进行泛化和二分化反应的分步协议。我们使用小鼠聚孔抑制复合物1(PRC1)、BMI1和RING1B(一种E3泛基质结合酶)的最小成分来说明这些反应,这种酶在119上具有单体蛋白H2A的一元组蛋白H2A。核糖体H2A的二次分体化是使用由人类二苯基辛酶BAP1和DEUbiquitinase ADaptor(DEUBAD)共同因子ASXL2的最小聚氨酯抑制双基酶(PR-DUB)复合物进行的。这些泛化/二氧化化测定可以在重组核小体与细菌纯化蛋白质重组或从哺乳动物细胞中纯化的原生核小体进行。我们强调能够对这些反应产生重大影响的复杂性,我们建议这些协议的一般原则可以迅速适应其他E3泛基苯丙酸酶和二苯基酶。

Introduction

泛化是最保守的翻译后修饰之一,对包括酵母、植物和脊椎动物在内的各种生物体至关重要。泛化包括泛黄蛋白的共价附着,一种高度保守的76个氨基酸多肽,以靶向蛋白质,并发生在三个连续步骤中,涉及三种酶,即E1-激活、E2-结合和E3联苯1, 2,3.这种翻译后修饰在广泛的生物过程中起着核心作用。事实上,提供反应特异性的E3利气,构成一个超级酶系,是泛性酶系统4、5、6中最丰富的酶。蛋白质泛化的下游影响取决于修饰的性质:单体化、多单体化以及线性或分枝多聚体化。单体基质化很少与蛋白酶体降解相关,但这种修饰涉及调解各种信号事件。多聚体化涉及泛基体分子本身中的N-端或赖塞林残留物,而多聚基蛋白的命运取决于泛化蛋白链延伸中涉及的残留物。人们早就知道,由流酶48的泛性基质介导的多聚体化诱导蛋白酶降解。相反,通过无物的以酶63进行多聚体化通常与蛋白质活化7、8、9相关。与其他重要的转化后修饰类似,泛化是可逆的,从蛋白质中去除泛化物通过称为二苯基苯酶(DUBs)的特定蛋白酶得到保证,这些蛋白酶已成为细胞过程的重要调节器2,10.重要的是,许多DUB是高度专业化的,并通过二次基质调节特定的基质,表明在泛化与二苯基化之间的良好平衡对蛋白质功能至关重要。E3s 和 DUB,以及蛋白酶体降解机制和附件因素,形成泛素蛋白酶体系统(UPS,具有 >1200 基因),用于调节主要信号通路,其中几个与细胞生长和增殖相关,细胞命运决定、分化、细胞迁移和细胞死亡。重要的是,放松管制的几个信号级联涉及泛化促进肿瘤发生和神经退化疾病5,11,12,13,14.

泛化在染色质生物学和DNA依赖过程15、16、17中起着普遍作用。例如,在以酶119(下文H2A K119ub)上单体化H2A是一种关键的转化后修饰,涉及转录抑制和DNA修复18、19、20 21,22.H2A K119ub 由 Polycomb 抑制复合体 1 (PRC1) 催化,在维护表观遗传信息方面发挥着关键作用,并且高度保存从果蝇到人类。规范PRC1是由RING1B和BMI1组成,它们是负责上述泛化事件22、23的核心E3泛基体。在果蝇中,H2A单体素(H2A K118ub,对应于哺乳动物中的H2A K119ub)由DUB Calypso逆转,它与附加性梳子(ASX)相互作用,形成聚氨酯抑制DUB(PR-DUB)复合体24。calypso的哺乳动物矫形原,BAP1,是一种肿瘤抑制器,在各种人类恶性肿瘤中被删除或灭活,25,26,27,28, 29,30,31,32,33. BAP1调节细胞核中的DNA依赖过程和内质性细胞凋亡33、34、35、3637,38,39,40,41,42. BAP1组装了多亚单位蛋白复合物,其中含有转录调节剂,特别是ASXL1、ASXL2和ASXL3(ASXLs),ASX38,43的三个正交。ASXL 使用 DEUBiquitinase ADaptor (DEUBAD) 域(也称为 ASXM 域)来刺激 BAP1DUB 活动 35、36、44 。因此,ASXL在协调染色质的BAP1 DUB活性和更广泛的肿瘤抑制功能方面发挥着重要作用。

存在几种研究泛化和二分化过程的方法。值得注意的是,使用从细菌中纯化的蛋白质进行生物化学检测在证明特定基质直接普及或去除泛化物方面仍然非常强大。这些实验可以进行,以调查一系列参数,如确定最小复合物的要求,确定反应动力学,定义结构/功能关系,以及了解病理的影响基因突变。在这里,我们提供协议,在染色质基质基材上进行泛化和二甲化反应,具有纯化成分。作为一种模型系统,提出了核糖体H2A蛋白的体外泛化和二次体化。以RING1B/BMI1和BAP1/DEUBAD的最小复合物组装的细菌纯化蛋白分别用于核细胞H2A的泛化或二聚位化。

Protocol

1. GSH-agarose 亲和力纯化 GST-RING1B (1-159)-BMI1 (1-109) E3 泛基丁利加酶复合物 使用 pGEX6p2rbs-GST-RING1B (1-159 aa) – BMI1 (1-109aa) 细菌表达结构来转化 BL21 (DE3) 细菌 (参见材料表)23。此构造允许将鼠圈 1B 域 1-159 融合到 BMI1 域 1-109,并在 pGEX-6P-2 主干中带有 GST 标记。 通过在存在 100 μg/mL 阿霉素和 50 μg/mL 氯霉素的情况下,在 20 mL 的 LB 溴介质中接种表达 GST-RING1B (1-159 aa) …

Representative Results

GST-BMI1和RING1B蛋白在细菌中生产良好,可轻易提取为可溶性成分。图 1A显示了用于典型纯化 GST-BMI1-RING1B 复合物的 Coomassie 蓝色染色。GST-BMI1 和 RING1B 波段以预期的分子量迁移,分别为 ±45 kDa 和 +13 kDa。值得注意的是,E3连带复合物高度均匀,细菌蛋白污染物和/或降解产物水平极低。此外,纯化复合物的化学测量是最佳的,因为当摩尔比为1:1时,GST-RING1B蛋白的带…

Discussion

建立健壮的体外泛化和二次成体测定对感兴趣的蛋白质有几个优点。这些测定可用于:(i) 建立最佳条件并定义这些反应的最低要求,(ii) 确定酶动力学和生化常数,(iii) 定义可能影响这些反应的辅助因子或抑制剂的作用,(iv)识别相互作用界面,(v) 测试人工或疾病相关突变的影响,(vi) 建立可进一步用于开发高通量化学筛选检测的测定条件。在这里,我们通过描述我们如何在核体基板上执行BMI1/RING1B介导的H2A…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

我们感谢戴安娜·阿贾乌德的技术援助。这项工作得到了加拿大自然科学和工程研究理事会(2015-2020年)、魁北克基因组(2016-2019年)和加拿大基因组(2016-2019年)的资助,获得E.B.A.E.B.A.的资助,是魁北克圣地基金会(FRQ-S)的高级学者。L.M.和N.S.K.拥有FRQ-S的博士学位奖学金。H.B拥有来自高等教育部和突尼斯科学研究部和科尔基金会的博士学位奖学金。

Materials

Amylose agarose beads New England Biolabs #E8021
Amicon Ultra 0.5 ml centrifugal filters 10K Sigma-Aldrich #UFC501096
Anti-H2AK119ub (H2Aub) Cell Signaling Technology #8240
Anti-Flag-agarose beads Sigma-Aldrich #A4596
Anti-protease cocktail Sigma-Aldrich #P8340
BL21 (DE3) CodonPlus-RIL bacteria Agilent technologies #230240
DMEM Wisent #319-005-CL
Empty chromatography column Biorad #731-1550
Flag peptide Sigma-Aldrich #F3290
GSH-agarose beads Sigma-Aldrich #G4510
HEK293T ATCC #CRL-3216
Imidazole Sigma-Aldrich #I5513
Micrococcal nuclease (MNase) Sigma-Aldrich #N3755
Ni-NTA agarose beads ThermoFisher Scientific #88221
N-methylmaleimide (NEM) Bioshop #ETM222
Pore syringe filter 0.45 μm Sarstedt #83.1826
Polyethylenimine (PEI) Polysciences Inc #23966-1
pGEX6p2rbs-GST-RING1B(1-159)-Bmi1(1-109) Addgene #63139
Ub Activating Enzyme (UBE1) Boston Biochem #E-305
UBCH5C (UBE2D3) Boston Biochem #E2-627
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Referencias

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Tags

In Vitro UbiquitinationDeubiquitinationNucleosomal HistonesPurified ComponentsUbiquitination MechanismsDeubiquitination MechanismsEnzyme MutationsHuman DiseaseHigh-throughput ScreeningSmall Molecule InhibitorsTherapeuticsBacteria LysisPMSFAntiprotease CocktailGSH BeadsChromatography ColumnGlutathioneHis-BAP1MBP-DEUBADNickel NTA BeadsImidazole

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Masclef, L., Maxime, U., Ahmed, O., Sen Nkwe, N., Barbour, H., Iannantuono, N. V., Boubekeur, A., Daou, S., Affar, E. B. In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones. J. Vis. Exp. (149), e59385, doi:10.3791/59385 (2019).
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