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Bioquímica

E3泛素连接酶在哺乳动物细胞裂解物中底物泛素化的评价

Published: May 10, 2022
doi:
10.3791/63561
, , ,
1Department of Genetic and Evolution,Federal University of São Carlos, 2Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto,University of São Paulo

Summary

我们为哺乳动物细胞中特定底物和E3泛素连接酶的泛素化测定提供了详细的方案。HEK293T细胞系用于蛋白质过表达，通过免疫沉淀从细胞裂解物中纯化多泛型底物，并在SDS-PAGE中分离。免疫印迹用于可视化这种翻译后修饰。

Abstract

泛型化是一种发生在真核细胞中的翻译后修饰，对几种生物学途径的调节至关重要，包括细胞存活、增殖和分化。这是一个可逆的过程，由泛素通过至少三种不同酶的级联反应与底物的共价连接组成，这些酶由E1（泛素活化酶），E2（泛素结合酶）和E3（泛素连接酶）组成。E3复合物在底物识别和泛素化中起着重要作用。这里描述了一种方案，用于评估哺乳动物细胞中的底物泛素化，使用编码所选底物的质粒，E3泛素连接酶和标记的泛素的瞬时共转染。在裂解之前，用蛋白酶体抑制剂MG132（羧苄氧基-亮氨酸-亮氨酸）处理转染的细胞，以避免底物蛋白酶体降解。此外，将细胞提取物提交到小规模免疫沉淀（IP）中以纯化多泛素底物，以便随后使用泛素标签的特异性抗体通过免疫印迹（WB）进行检测。因此，描述了哺乳动物细胞中泛素化测定的一致且简单的方案，以帮助科学家解决特定底物和E3泛素连接酶的泛素化。

Introduction

翻译后修饰（PTMs）是关于蛋白质调控的重要机制，对细胞稳态至关重要。蛋白质泛素化是一种动态且复杂的修饰，可产生各种不同的信号，从而在真核生物中产生多种细胞结果。泛素化是一个可逆的过程，包括将含有76个氨基酸的泛素蛋白附着到底物上，发生在由三个不同反应组成的酶级联反应1中。第一步的特征在于泛素活化，这取决于ATP水解在泛素C端和存在于E1酶活性位点中的半胱氨酸残基之间形成高能硫酯连接的泛素。随后，泛素被转移到E2酶中，与泛素形成硫酯样复合物。之后，泛素通过E2共价连接到底物上，或者更常见的是E3酶，其识别并与底物2，3相互作用。偶尔，E4酶（泛素链伸长因子）对于促进多泛素链组装是必需的3。

泛素有七个赖氨酸残基（K6，K11，K27，K29，K33，K48和K63），允许形成多泛素链，产生不同的连锁反应，产生不同的三维结构，这些结构将被几种效应蛋白4，5识别。因此，在底物中引入的多泛素链对于决定其细胞命运6，7，8至关重要。此外，底物也可以通过其称为N-德隆的N端残基被泛素化。特定的E3泛素连接酶负责N-degron识别，允许附近赖氨酸残基的多泛素化9。

如今，有40多种不同的SCF专用基板被表征。其中，几种生物途径的关键调节因子，包括细胞分化和发育以及细胞存活和死亡，可以找到10，11，12，13。因此，鉴定每个E3泛素连接酶的特定底物对于设计各种生物事件的综合图谱至关重要。尽管鉴定真正的底物在生化上具有挑战性，但使用基于生物化学的方法非常适合评估链特异性以及单泛型和多泛型化之间的区别14。本研究描述了使用哺乳动物细胞系HEK293T过表达底物UXT-V2（普遍表达的预折叠样伴侣同种型2）与E3泛素 – 连接酶复合物SCF（Fbxo7）的泛素化测定的完整方案。UXT-V2是NF-κB信号传导的重要辅助因子，一旦这种蛋白质在细胞中被敲低，它就会抑制TNF-α诱导的NF-κB活化11。因此，为了检测多泛型UXT-V2，使用蛋白酶体抑制剂MG132，因为它具有阻断蛋白酶体复合物15的26S亚基的蛋白水解活性的能力。此外，将细胞提取物提交给小规模IP以纯化底物，利用固定在琼脂糖树脂中的特异性抗体，随后由WB使用选定的抗体进行检测。该协议对于验证细胞环境中的底物泛素化非常有用，并且还可以适应不同类型的哺乳动物细胞和其他E3泛素 – 连接酶复合物。然而，有必要验证通过 体外 泛型化测定测试的底物，因为两种方案在鉴定真实底物方面相互补充。

Protocol

注意： 图1概述了哺乳动物细胞中的泛型化测定方案。 图 1. 泛素化测定程序概述。 请点击此处查看此图的大图。 1. 细胞培养…

Representative Results

UXT（普遍表达的转录本）是一种前折叠蛋白样蛋白，在小鼠和人体组织（如心脏、大脑、骨骼肌、胎盘、胰腺、肾脏和肝脏）中形成普遍表达的蛋白质折叠复合物18.已经描述了两种名为UXT-V1和UXT-V2的UXT剪接同种型，它们执行不同的功能和亚细胞位置。UXT-V1主要局限于细胞质和线粒体内，并且与TNF-α诱导的细胞凋亡和抗病毒信号体形成19，20<…

Discussion

泛型化是一种基本的翻译后修饰，可调节几种蛋白质的水平，并在许多信号通路和生物过程中起着至关重要的作用，从而确保健康的细胞内环境。泛素-蛋白酶体系统（UPS）是最近药物研究的主要焦点之一，它提供了稳定肿瘤抑制因子或诱导致癌产物降解的可能性22。例如，在多发性骨髓瘤 （MM） 中负责单克隆免疫球蛋白分泌的浆细胞肿瘤的异常增殖促进了病理生理学途径，这是…

Declarações

The authors have nothing to disclose.

Acknowledgements

F.R.T 由 FAPESP 授权号 2020/15771-6 和 CNPq 通用 405836/2018-0 提供支持。公共战略计划和 V.S 由资本保护计划提供支持。C.R.S.T.B.C.获得了FAPESP奖学金编号2019 / 23466-1的支持。我们感谢桑德拉·丸山（FAPESP 2016/20258-0）的物质支持。

Materials

1.5 mL microtube Axygen PMI110-06A
100 mm TC-treated culture dish Corning 430167
15 mL tube Corning 430766
96-well plate Cralplast 655111
Agarose-anti-HA beads Sigma-Aldrich E6779
Anti Mouse antibody Seracare 5220-0341 Goat anti-Mouse IgG
Anti Rabbit antibody Seracare 5220-0337 Goat anti-Rabbit IgG
Anti-Actin antibody Sigma-Aldrich A3853 Dilution used: 1:2000
Anti-Fbxo7 antibody Sigma-Aldrich SAB1407251 Dilution used: 1:1000
Anti-HA antibody Sigma-Aldrich H3663 Dilution used: 1:1000
Anti-Myc antibody Cell Signalling 2272 Dilution used: 1:1000
Bradford reagent Sigma-Aldrich B6916-500ML
BSA Sigma-Aldrich A9647-100G Bovine Serum Albumin
Cell incubator Nuaire NU-4850
Centrifuge Eppendorf 5804R 500 x g for 5 min
ChemiDoc BioRad
Digital pH meter Kasvi K39-2014B
Dulbecco’s Modified Eagle’s Medium Corning 10-017-CRV High glucose
Fetal bovine serum Gibco F4135 Filtrate prior use
HA peptide Sigma-Aldrich I2149
HEK293T cells ATCC CRL-3216
Hepes Gibco 15630080
KCl VWR Life Science 0365-500G
Kline rotator Global Trade Technology GT-2OIBD
MG-132 Boston Biochem I-130
Microcentrifuge Eppendorf 5418R
Na3VO4 (Ortovanadato)
NaF
Nitrocellulose blotting membrane GE Healthcare 10600016
NP40 (IGEPAL CA-630) Sigma-Aldrich I8896-100ML
Optical microscope OPTIKA microscopes SN510768
Opti-MEM Gibco 31985-070
pcDNA3 Invitrogen V79020 For mammalian expression
pcDNA3-2xFlag-Fbxo7  Kindly donated by Dr. Marcelo Damário Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI
pcDNA3-2xFlag-Fbxo7-ΔF-box  Kindly donated by Dr. Marcelo Damário Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI. Δ335-367
pcDNA3-UXTV2-HA  Kindly donated by Dr. Marcelo Damário Tag HA (C-terminal). Restriction enzymes: EcoRI and XhoI
pCMV-6xHis-Myc-Ubiquitin  Kindly donated by Dr. Marcelo Damário Tag 6x-His-Myc (N-terminal). Restriction enzymes: EcoRI and KpnI
Pen Strep Glutamine 100x Gibco 10378-016
Phosphate buffered saline 10x AccuGENE 51226 To obtain a 1x PBS, dilute the 10x PBS into ultrapure water
Polyethylenimine (PEI) Sigma-Aldrich 9002-98-6
Ponceau S VWR Life Science 0860-50G
Protease inhibitor cocktail SIGMAFAST Sigma-Aldrich S8820
Rocking Shaker Kasvi 19010005
SDS-PAGE system BioRad 165-8004
Solution Homogenizer Phoenix Luferco AP-22
Trizma base Sigma-Aldrich T6066-500G
Trypsine (TrypLe Express) Gibco 12605-028
Western Blotting Luminol Reagent Santa Cruz Biotechnology SC-2048
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Tags

Keyword Extraction: Substrate UbiquitylationE3 LigaseMammalian Cell LysatesImmunoprecipitation AssayWestern BlotHuman DiseasesCancerNeurological DisordersE3 Ligase DysregulationHEK293T Cell LineDulbecco’s Modified Eagles MediumFetal Bovine SerumPenicillinStreptomycinL-glutamineTrypsin EDTAPolyethyleneimineOpti-MEM 1 Reduced Serum MediumTransient Transfections
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dos Passos, P. M. S., Spagnol, V., de Correia, C. R., Teixeira, F. R. Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates. J. Vis. Exp. (183), e63561, doi:10.3791/63561 (2022).
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