Name: in vitro ubiquitination and deubiquitination assays of nucleosomal histones
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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。

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
<ol><li>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。</li>
	<li>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) ...

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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...

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。

<table>
	<tbody>
		<tr>
			<td>Amylose agarose beads</td>
			<td>New England Biolabs</td>
			<td>#E8021</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 ml centrifugal filters 10K</td>
			<td>Sigma-Aldrich</td>
			<td>#UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-H2AK119ub (H2Aub)</td>
			<td>Cell Signaling Technology</td>
			<td>#8240</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Flag-agarose beads</td>
			<td>Sigma-Aldrich</td>
			<td>#A4596</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-protease cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>#P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) CodonPlus-RIL bacteria</td>
			<td>Agilent technologies</td>
			<td>#230240</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Wisent</td>
			<td>#319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Empty chromatography column</td>
			<td>Biorad</td>
			<td>#731-1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Flag peptide</td>
			<td>Sigma-Aldrich</td>
			<td>#F3290</td>
			<td></td>
		</tr>
		<tr>
			<td>GSH-agarose beads</td>
			<td>Sigma-Aldrich</td>
			<td>#G4510</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T</td>
			<td>ATCC</td>
			<td>#CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>#I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Micrococcal nuclease (MNase)</td>
			<td>Sigma-Aldrich</td>
			<td>#N3755</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA agarose beads</td>
			<td>ThermoFisher Scientific</td>
			<td>#88221</td>
			<td></td>
		</tr>
		<tr>
			<td>N-methylmaleimide (NEM)</td>
			<td>Bioshop</td>
			<td>#ETM222</td>
			<td></td>
		</tr>
		<tr>
			<td>Pore syringe filter 0.45 &#956;m</td>
			<td>Sarstedt</td>
			<td>#83.1826</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>Polysciences Inc</td>
			<td>#23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX6p2rbs-GST-RING1B(1-159)-Bmi1(1-109)</td>
			<td>Addgene</td>
			<td>#63139</td>
			<td></td>
		</tr>
		<tr>
			<td>Ub Activating Enzyme (UBE1)</td>
			<td>Boston Biochem</td>
			<td>#E-305</td>
			<td></td>
		</tr>
		<tr>
			<td>UBCH5C (UBE2D3)</td>
			<td>Boston Biochem</td>
			<td>#E2-627</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro ubiquitination and deubiquitination assays of nucleosomal histones

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。

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...

Watch this Scientific Journal Video about In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones at JoVE.com

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

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。

核糖体组蛋白的体外泛化和二次基质测定

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

鉴于细胞内泛化和脱归反应的复杂性，使用定义的纯化成分建立体外测定非常重要。这有助于理解泛化和脱泛化的机制，并确定与编码负责这种反应的酶的基因相关的突变如何导致人类疾病。我们可以识别驱动这些反应的这些组件中的域和图案。我们还可以开发高通量的屏幕检测，可用于筛选小分子抑制剂，这些抑制剂可以开发为潜在的治疗方法。要开始细菌解菌，将PMSF和抗蛋白鸡尾酒加入冰冷解液缓冲液 A.将25毫升的解菌缓冲液 A加入含有细菌颗粒的瓶子中，然后重新释放颗粒。将含有细菌的利酸盐转移到管中进行声波化。使用探针声波器以 70%输出振幅对酸盐进行声波化，30 秒，4 到 5 次。然后在摄氏4度下以21，000次g离心20分钟。根据手稿，将细菌与GSH珠子分离5小时后，将珠子连同10毫微缓冲液一起转移到空色谱柱中。然后，将含有25毫毛托美的1.5毫安第安进柱中，通过重力将洗脱收集到1.5毫升微管中。使用新鲜缓冲器重复洗脱过程四次 A 根据手稿继续处理洗脱分数。首先，在25毫升的冰冷解液缓冲液B.Mix中重新填充Sss-BAP1和MBP-DEUBAD细菌颗粒，并在冰上孵育15分钟。声波化，然后以21，000倍g离心20分钟。离化离心后，通过0.45微米孔注射器过滤器过滤回乳，放入瓶子中，与镍NTA珠混合。在4摄氏度下孵育，摇晃5小时。之后，以25，000次g旋转珠子三分钟。使用移液器将上经器保存在 50 毫升管中，以流经。用5毫微缓冲B洗涤珠子，含有20毫摩尔，用3分钟离心机洗六次。上次洗涤后，将含有 10 毫微缓冲液 B 的珠子转移到空色谱柱中，并加入含有 200 毫摩尔 imidazole 的一毫升缓冲液 B。将重力洗脱收集成含有DTT和EDTA的1.5毫升微管。重复洗脱步骤四次，将所需的洗脱分数汇集到 15 毫升管中。添加缓冲区 C 以稀释洗小的复合物三次。然后将稀释的洗脱转移到管中准备好的阿米洛塞珠中，并在四摄氏度下孵育，并摇动。早晨，将管以2，500次g离心三分钟，将上经液留在15毫升管中，作为流经。用五毫的缓冲C清洗蛋白质结合的珠子五至六次。然后将500微升缓冲器D添加到紫化的Ss-BAP1/MBP-DEUBAD复合物上，在阿米洛塞阿加罗斯珠上制造50%珠子溶液。将 50% 珠子溶液的 20 微升转移到微离心管中，并添加 20 微升的 2 倍 Laemmli 样品缓冲液，用于 SDS-PAGE 和库马西蓝色分析。将剩余的珠子溶液存放在零下 80 摄氏度，供将来使用。在培养 HEK293T 细胞后，在 15 厘米培养皿中，在转染前一天，在 20 毫微全培养基中培养 1200 万个细胞。一天后，在细胞培养罩下，吸气培养素，并供应12毫微的血清自由培养素。使用63微升PEI以每毫升1毫克的速度，用21微克的pCDNA平-H2A对细胞进行转染。12 小时后，更改为完成介质。根据手稿采集细胞后，将细胞颗粒重新填充在约10卷含有NEM的缓冲液E中，以抑制脱皮酶，并在冰上孵育20分钟。在3000次g下离心5分钟后，丢弃上经剂。先用10卷缓冲液E洗两次染色质颗粒，然后用缓冲液F.用5毫微量的缓冲液中重新暂停染色质，并在室温下以每毫升3个单位的浓度处理微球蛋白酶，10分钟。孵育后，将混合物的40微升等分转移到1.5毫升管中，以分析核糖体DNA片段。加入40微升苯酚氯仿和20微升6倍DNA加载缓冲液。漩涡和旋转在18，000次g两分钟。然后在2%的加糖凝胶中转移水相的15微升，以加载DNA。根据手稿停止反应和离心后，在4摄氏度的15毫升管中用抗旗树脂珠孵育可溶性染色质分数，并摇动。用缓冲器 G 洗六次珠子，用五毫的缓冲 G 将珠子转移到空色谱柱中。用260微升缓冲G稀释珠子的束缚核糖体，每毫升旗洗肽含有200微克，在pH8下含有1至5个摩尔三叶草。将核糖体吸三次，在寒冷的房间里一小时。为了进行泛化测定，首先在40微升缓冲液H中加入一微克纯化核糖体，然后加入含有250南片的UB1、672纳米的UB和E2 UB结合酶和1微克BB1-1B-1B E3泛素连结酶复合物的酶溶液混合物。在37摄氏度下孵育反应3小时，偶尔摇晃。通过添加 40 微升两次 Laemmli 样品缓冲液，并分析西印的组蛋白 H2AK119 泛光，从而停止反应。进行体外脱比化测定，在40微升缓冲液一和1微克细菌纯化的Ss-BAP1/MBP-DEUBAD珠体中重新填充纯化的核糖体。将管子放在37摄氏度的培养箱中3小时，进行偶尔摇晃的脱泛反应。然后通过添加40微升两倍莱姆利缓冲液，通过免疫印迹进行分析来停止反应。在这个实验中，使用GST亲和珠对GST-BM1-RING1B复合物的典型纯化进行库马西蓝色染色表明，波段在预期摩尔重量下迁移，分别约为45千达吨和13千陀吨。同样，His-BAP1 MBP-DEUBAD复合物的纯化也导致相对高的同质制剂，带子分别约为90千吨和55千元。另一方面，纯化核子体分数基本上由147基对带组成，表明存在高度富集的单核细胞分。纯核素的库马西蓝色染色显示了四个组蛋白亚单位的典型波段模式，具有计量量。组蛋白H2A与BMI1-RING1B复合物的泛化表明蛋白质泛素形式的时间依赖性增加，而非MODATED形式的水平同时降低。泛向反应是总的，因为几乎所有的H2A蛋白都转化为H2A K119ub。另一方面，对本土核糖体进行了非泛化测定。这些核糖体与BAP1-DEUBAD孵育后，观察到H2A K119ub信号的时间依赖性减少。泛化和去泛化反应的成功取决于蛋白质的纯化方式。我们需要良好的产量，蛋白质再依赖和纯化的缓冲液应与生化反应相容。当这种泛化和去泛化检测已经建立，我们可以测试影响激活剂和抑制剂，以及疾病相关基因突变的影响。该方法由几个实验室的贡献建立，对了解泛化和非泛化机制非常有用。几种化学品是危险的，需要在发动机罩中操作。这包括苯酚氯仿、酸、碱和还原剂。

Research

JoVE Journal

Biochemistry

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

自动声胶肽受体激动剂的效价测定测定的连续稀释

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

科研

生物化学

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

核小体的重建与差异同位素标记的姐妹组蛋白

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

用天然电泳，凝胶电泳和电解法分析线粒体电子传递链的超复合物

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Quantification of Hypopigmentation Activity In Vitro

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

生理活性类囊体的分离及其在能量依赖性蛋白转运检测中的应用

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

RING-E3型无二体气体在Vitro和Planta中的功能特性

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

基于无标签阻抗的 GPCR 检测中用于提高吞吐量的逐步加量协议

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...